Dermatophagoides nucleic acid molecules, proteins and uses thereof

ABSTRACT

The present invention relates to high molecular weight Dermatophagoides proteins, nucleic acid molecules encoding such proteins, and therapeutic and diagnostic reagents derived from such proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to prior U.S. ProvisionalApplication Serial No. 60/098,909, filed Sep. 2, 1998, now abandonedentitled “NOVEL DERMATOPHAGOIDES NUCLEIC ACID MOLECULES, PROTEINS ANDUSES THEREOF”; U.S. Provisional Application Serial No. 60/085,295, filedMay 13, 1998, entitled “NOVEL DERMATOPHAGOIDES PROTEINS AND USESTHEREOF”; and U.S. application Ser. No. 09/062,013, filed Apr. 17, 1998,converted by Petition on May 13, 1998 to U.S. Provisional ApplicationSerial No. 60/098,565, entitled “NOVEL DERMATOPHAGOIDES PROTEINS ANDUSES THEREOF”; each of which is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to high molecular weight Dermatophagoidesproteins, nucleic acid molecules and therapeutic and diagnostic reagentsderived from such proteins.

BACKGROUND OF THE INVENTION

Immunoglobulin E (IgE) mediated allergic symptoms afflict many animals.IgE antibody production in an animal can induce pathogenic IgE responsesincluding, for example, atopic disease, asthma and rhinitis. Allergensare proteins or peptides characterized by their ability to induce apathogenic IgE response in susceptible individuals.

House dust mite (e.g., Dermatophagoides farinae and Dermatophagoidespteronyssinus; Der f and Der p, respectively) allergens are majorcausative agents associated with IgE-mediated pathogenesis. Previousinvestigators have identified two major groups of dust mite allergens inhumans, group I (Der fI and Der p I, Mr 25,000) and group 2 (Der fII andDer p II, Mr 14,000); reviewed in Chapman, et al., Allergy, vol. 52,pp.37-379, 1997. Prior investigators have disclosed nucleotide and/oramino acid sequences for: Der fI, Der fII, Der p I and Der p II, U.S.Pat. No. 5,552,142, to Thomas et al., issued Sep. 3, 1996, U.S. Pat. No.5,460,977, to Ando et al., issued Oct. 24, 1995, PCT Patent PublicationNo. WO 95/28424, by Chen et al., published Oct. 26, 1995, U.S. Pat. No.5,433,948, to Thomas et al., issued Jul. 18, 1995, PCT PatentPublication No. WO 93/08279, by Garmen et al., published Mar. 4, 1993,or Chapman, ibid.; Derp III, PCT Patent Publication No. WO 95/15976, byThomas et al., published Jun. 15, 1995; Der p VII, PCT PatentPublication No. WO 94/20614, by Thomas et al., published Sep. 15, 1994;a 40-kilodalton (kd) Der f allergen, U.S. Pat. No. 5,405,758, to Oka etal., issued Apr. 11, 1995, U.S. Pat. No. 5,314,991, to Oka et al.,issued May 24, 1994; a 70-kd Der f allergen which is a heat shockprotein (Hsp70), Aki et al., J. Biochem., vol. 115, pp. 435-440, 1994;or Noli et al., Vet. Immunol. Immunopath., vol. 52, pp. 147-157, 1996;and a 98-kd Der f paramyosin-like allergen, Tsai et al, J. Allergy Clin.Immunol., vol. 102, pp. 295-303, 1998. None of these published sequencesindicates, suggests or predicts any of the mite allergic nucleic acidmolecules or proteins of the present invention, nor the relevance ofsuch proteins as being immunoreactive with IgE antibodies in canine,feline, or human sera.

Products and processes of the present invention are needed in the artthat provide specific detection and treatment of mite allergy.

SUMMARY OF THE INVENTION

The present invention relates to novel proteins having molecular weightsof about 60 kilodaltons (kd or kD), 70 kD, or from about 98 kD to about109 kD. Such proteins include at least one epitope of a protein allergenof a mite of the genus Dermatophagoides and are designated herein as DerHMW-map proteins. Preferred proteins are Dermatophagoides farinae orDermatophagoides pteronyssius proteins. The present invention alsoprovides proteins that are fragments or peptides of full-length ormature proteins, as well as antibodies, mimetopes or muteins of any ofsuch proteins. The present invention also provides nucleic acidmolecules encoding any of such proteins, as well as complements thereof.The present invention also includes methods to obtain such proteins,nucleic acid molecules, antibodies, mimetopes or muteins, as well asmethods to use such compounds in diagnostic or therapeutic applications.The present invention also relates to reagents comprisingnon-proteinaceous epitopes that bind to IgE in mite-allergic dogs and/orcats as well as to antibodies raised against such epitopes. The presentinvention also relates to therapeutic compositions or assay kitscomprising such non-proteinaceous epitopes, as well as to methods toidentify and/or desensitize an animal susceptible to an allergicresponse to a mite, comprising the use of non-proteinaceous epitopes ofthe present invention.

One embodiment of the present invention is at least one of the followingisolated nucleic acid molecules: (a) a nucleic acid molecule comprisingat least about 150 nucleotides, wherein such a nucleic acid moleculehybridizes, in a solution comprising 1×SSC and 0% formamide, at atemperature of about 50° C., to a nucleic acid molecule comprising atleast one of the following nucleic acid sequences: SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ IDNO:42, SEQ ID NO:43, SEQ ID NO:45, and a nucleic acid sequence encodinga protein comprising the amino acid sequence of SEQ ID NO:33 and acomplement thereof; and (b) a nucleic acid molecule comprising afragment of any of the nucleic acid molecules of (a) wherein thefragment comprises at least about 15 nucleotides. The present inventionalso includes recombinant molecules, recombinant viruses and recombinantcells comprising such nucleic acid sequences as well as methods toproduce them.

Another embodiment of the present invention is an isolated proteinencoded by at least one of the following nucleic acid molecules: (a) anucleic acid molecule comprising at least about 150 nucleotides, whereinsuch a nucleic acid molecule hybridizes, in a solution comprising 1×SSCand 0% formamide, at a temperature of about 50° C., to a nucleic acidmolecule comprising at least one of the following nucleic acidsequences: SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:36, SEQID NO:39, SEQ ID NO:42, SEQ ID NO:45, and a complement of a nucleic acidsequence encoding a protein comprising the amino acid sequence SEQ IDNO:33; and (b) a nucleic acid molecule comprising a fragment of any ofthe nucleic acid molecules of (a), wherein the fragment comprises atleast about 15 nucleotides. An isolated protein of the present inventioncan also be encoded by a nucleic acid molecule that hybridizes understringent hybridization conditions with the complement of a nucleic acidmolecule that encodes a protein having at least one of the followingamino acid sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15,SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35,SEQ ID NO:38, SEQ ID NO:41, and SEQ ID NO:44. The present invention alsoincludes an antibody that selectively binds to a protein of the presentinvention as well as methods to produce and use such proteins orantibodies.

The present invention also includes a therapeutic composition fortreating an allergic response to a mite. Such a therapeutic compositionincludes at least one of the following desensitizing compounds: (a) anisolated nucleic acid molecule of the present invention; (b) an isolatedmite allergenic protein of the present invention; (c) a mimetope of sucha mite allergenic protein; (d) a mutein of such a mite allergenicprotein; (e) an antibody to such a mite allergic protein; and (f) aninhibitor of binding of such a mite allergic protein to IgE. Alsoincluded is a method to desensitize a host animal to an allergicresponse to a mite. Such a method includes the step of administering tothe animal a therapeutic composition of the present invention.

One embodiment of the present invention is an assay kit for testing ifan animal is susceptible to or has an allergic response to a mite. Sucha kit includes an isolated protein of the present invention and a meansfor determining if the animal is susceptible to or has that allergicresponse. Such a means includes use of such a protein to identifyanimals susceptible to or having allergic responses to mites. Thepresent invention also includes a method to identify an animalsusceptible to or having an allergic response to a mite. Such a methodincludes the steps of: (a) contacting an isolated protein of the presentinvention with antibodies of an animal; and (b) determiningimmunocomplex formation between the protein and the antibodies, whereinformation of the immunocomplex indicates that the animal is susceptibleto or has such an allergic response.

The present invention includes a reagent that comprises anon-proteinaceous epitope having at least one of the followingidentifying characteristics: (a) the epitope is resistant toβ-elimination of peptides; (b) the epitope is resistant to Proteinase-Kdigestion; and (c) the epitope is reactive to a test designed to detectglycosylated proteins. Such an epitope binds to at least one of thefollowing antibodies: canine IgE from dogs allergic to mites and felineIgE from cats allergic to mites. Also included is an isolated antibodythat selectively binds such a non-proteinaceous epitope as well asderivatives of such an epitope.

The present invention also relates to therapeutic compositions and assaykits comprising a non-proteinaceous epitope of the present invention, aswell as methods to identify and/or desensitize an animal susceptible toan allergic response to a mite, comprising the use of anon-proteinaceous epitope of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates high molecular weight Der f proteins resolved by 12%Tris-Glycine SDS-PAGE.

FIG. 2 illustrates an about 60 kD Der f protein resolved by 14%Tris-Glycine SDS-PAGE.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for isolated proteins having molecularweights ranging from about 60 kilodaltons (kD) to about 109 kD, thatinclude at least one epitope of a protein allergen of a mite of thegenus Dermatophagoides, in particular a mite of the speciesDermatophagoides farinae and/or Dermatophagoides pteronyssius. Suchproteins are referred to herein as Der HMW-map proteins. The presentinvention further includes methods to isolate and identify nucleic acidmolecules encoding Der HMW-map proteins, antibodies directed against DerHMW-map proteins and inhibitors of Der HMW-map protein activity. As usedherein, the term isolated Der HMW-map proteins refers to Der HMW-mapproteins derived from Dermatophagoides, and more preferably fromDermatophagoides farinae and/or Dermatophagoides pteronyssius and, assuch, can be obtained from its natural source or can be produced using,for example, recombinant nucleic acid technology or chemical synthesis.Also included in the present invention is the use of this protein andantibodies in a method to detect immunoglobulin that specifically bindsto Der HMW-map proteins, to treat pathogenesis against mite allergens,and in other applications, such as those disclosed below. The productsand processes of the present invention are advantageous because theyenable the detection of anti-Der HMW-map antibodies in fluids of animalsand the inhibition of IgE or Der HMW-map protein activity associatedwith disease.

One embodiment of the present invention is an isolated Dermatophagoidesallergenic composition including: (a) a composition produced by a methodcomprising: (1) applying soluble proteins of a Dermatophagoides extractto a gel filtration column; (2) collecting excluded protein from the gelfiltration column and applying the excluded protein to an anion exchangecolumn; and (3) eluting proteins bound to the anion exchange column withabout 0.3 M Tris-HCl, pH 8 to obtain the Dermatophagoides allergeniccomposition; and (b) a composition comprising a peptide of a proteinproduced in accordance with step (a), in which the allergeniccomposition is capable of a biological function including binding toIgE, stimulating a B lymphocyte response and stimulating a T lymphocyteresponse. Such Dermatophagoides allergenic composition is also referredto herein as a Der HMW-map composition. A suitable gel filtration columnincludes any gel filtration column capable of excluding proteins havinga molecular weight between about 50 kD and about 150 kD. A preferred gelfiltration column includes, but is not limited to a Sephacryl S-100column. A suitable anion exchange column includes any anion exchangecolumn capable of binding to a protein having a pI of less than about pI6. A preferred anion exchange column includes, but is not limited to aQ-Sepharose column. As used herein, “stimulating a B lymphocyteresponse” refers to increasing a humoral immune response in an animalthat is induced preferentially by a Der HMW-map of the present inventionand involves the activity of a B lymphocyte in the animal. As usedherein, “stimulating a T lymphocyte response” refers to increasing acellular immune response in an animal that is induced preferentially bya Der HMW-map of the present invention and involves the activity of a Tlymphocyte in the animal.

One embodiment of the present invention is an isolated protein thatincludes a Der HMW-map protein. It is to be noted that the term “a” or“an” entity refers to one or more of that entity; for example, aprotein, a nucleic acid molecule, an antibody, an inhibitor, a compoundor a therapeutic composition refers to “one or more” or “at least one”protein, nucleic acid molecule, antibody, inhibitor, compound ortherapeutic composition respectively. As such, the terms “a” (or “an”),“one or more” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprising”, “including”, and“having” can be used interchangeably. According to the presentinvention, an isolated, or biologically pure, protein, is a protein thathas been removed from its natural milieu. As such, “isolated” and“biologically pure” do not necessarily reflect the extent to which theprotein has been purified. An isolated protein of the present inventioncan be obtained from its natural source, can be produced usingrecombinant DNA technology, or can be produced by chemical synthesis.

As used herein, a Der HMW-map protein can be a full-length protein orany homolog of such a protein. As used herein, a protein can be apolypeptide or a peptide, as the terms are used by those of skill in theart. Preferably, a Der HMW-map protein comprises at least a portion of aDer HMW-map protein that comprises at least one epitope recognized by anIgE antibody (i.e., a protein of the present invention binds to an IgEantibody), an antibody on the surface of a B lymphocyte and/or a T-cellreceptor in the presence of a major histocompatability complex (MHC)molecule from an animal demonstrating IgE-mediated pathogenesis to a DerHMW-map protein.

A peptide of the present invention includes a Der HMW-map protein of thepresent invention that is capable of binding to IgE, desensitizing ananimal against mite allergen, stimulating a B lymphocyte response,and/or stimulating a T lymphocyte response. Preferably, a peptide of thepresent invention comprises a B lymphocyte epitope or a T lymphocyteepitope. A peptide having a B lymphocyte epitope can bind to anantibody. A peptide having a T lymphocyte epitope can bind to a MHCmolecule in such a manner that the peptide can stimulate a T lymphocytethrough a T cell receptor. According to the present invention, a peptidecomprising a B lymphocyte epitope can be from about 4 residues to about50 residues in length, preferably from about 5 residues to about 20residues in length. According to the present invention, a peptidecomprising a T lymphocyte epitope can be from about 4 residues to about20 residues in length, preferably from about 8 residues to about 16residues in length.

A Der HMW-map protein of the present invention, including a homolog, canbe identified in a straight-forward manner by the protein's ability toinduce an allergic response to Der HMW-map protein. Examples of DerHMW-map protein homologs include Der HMW-map protein in which aminoacids have been deleted (e.g., a truncated version of the protein, suchas a peptide), inserted, inverted, substituted and/or derivatized (e.g.,by glycosylation, phosphorylation, acetylation, myristoylation,prenylation, palmitoylation, amidation and/or addition ofglycerophosphatidyl inositol) such that the homolog is capable ofinducing an allergic response to a natural Der HMW-map protein.

Der HMW-map protein homologs can be the result of natural allelicvariation or natural mutation. Der HMW-map protein homologs of thepresent invention can also be produced using techniques known in the artincluding, but not limited to, direct modifications to the protein ormodifications to the gene encoding the protein using, for example,classic or recombinant nucleic acid techniques to effect random ortargeted mutagenesis.

One embodiment of the present invention is a Der HMW-map gene thatincludes the nucleic acid sequence SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:20 SEQ ID NO:22, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:43, and SEQ ID NO:45 as well as the complements of any of thesenucleic acid sequences. These nucleic acid sequences are furtherdescribed herein. For example, nucleic acid sequence SEQ ID NO:14represents the deduced sequence of the coding strand of a cDNA(complementary DNA) denoted herein as Der HMW-map gene nucleic acidmolecule nDerf98₁₇₅₂, the production of which is disclosed in theExamples. Nucleic acid molecule nDerf98₁₇₅₂ comprises an apparentlyfull-length coding region. The complement of SEQ ID NO:14 (representedherein by SEQ ID NO:16) refers to the nucleic acid sequence of thestrand complementary to the strand having SEQ ID NO:14, which can easilybe determined by those skilled in the art. Likewise, a nucleic acidsequence complement of any nucleic acid sequence of the presentinvention refers to the nucleic acid sequence of the nucleic acid strandthat is complementary to (i.e., can form a double helix with) the strandfor which the sequence is cited. It should be noted that since nucleicacid sequencing technology is not entirely error-free, SEQ ID NO:14 (aswell as other nucleic acid and protein sequences presented herein)represents an apparent nucleic acid sequence of the nucleic acidmolecule encoding a Der HMW-map protein of the present invention.

In another embodiment, a Der HMW-map gene or nucleic acid molecule canbe an allelic variant that includes a similar but not identical sequenceto SEQ ID NO:14 or SEQ ID NO:16, or any other Der HMW-map nucleic acidsequence cited herein. For example, an allelic variant of a Der HMW-mapgene including SEQ ID NO:14 or SEQ ID NO:16, is a gene that occurs atessentially the same locus (or loci) in the genome as the gene includingSEQ ID NO:14 and SEQ ID NO:16, but which, due to natural variationscaused by, for example, mutation or recombination, has a similar but notidentical sequence. Because natural selection typically selects againstalterations that affect function, allelic variants (i.e. allelescorresponding to, or of, cited nucleic acid sequences) usually encodeproteins having similar activity to that of the protein encoded by thegene to which they are being compared. Allelic variants of genes ornucleic acid molecules can also comprise alterations in the 5′ or 3′untranslated regions of the gene (e.g., in regulatory control regions),or can involve alternative splicing of a nascent transcript, therebybringing alternative exons into juxtaposition. Allelic variants are wellknown to those skilled in the art and would be expected to occurnaturally within a given dust mite such as Dermatophagoides, since therespective genomes are diploid, and sexual reproduction will result inthe reassortment of alleles.

In one embodiment of the present invention, an isolated Der HMW-mapprotein is encoded by a nucleic acid molecule that hybridizes understringent hybridization conditions to a gene encoding a Der HMW-mapprotein. The minimal size of a Der HMW-map protein of the presentinvention is a size sufficient to be encoded by a nucleic acid moleculecapable of forming a stable hybrid (i.e., hybridizing under stringenthybridization conditions) with the complementary sequence of a nucleicacid molecule encoding the corresponding natural protein. The size of anucleic acid molecule encoding such a protein is dependent on thenucleic acid composition and the percent homology between the DerHMW-map nucleic acid molecule and the complementary nucleic acidsequence. It can easily be understood that the extent of homologyrequired to form a stable hybrid under stringent conditions can varydepending on whether the homologous sequences are interspersedthroughout a given nucleic acid molecule or are clustered (i.e.,localized) in distinct regions on a given nucleic acid molecule.

The minimal size of a nucleic acid molecule capable of forming a stablehybrid with a gene encoding a Der HMW-map protein is typically at leastabout 12 nucleotides to about 15 nucleotides in length if the nucleicacid molecule is GC-rich and at least about 15 to about 17 bases inlength if it is AT-rich. The minimal size of a nucleic acid moleculeused to encode a Der HMW-map protein homolog of the present invention isfrom about 12 to about 18 nucleotides in length, preferably about 12nucleotides, or about 15 nucleotides, or about 18 nucleotides in length.Thus, the minimal size of a Der HMW-map protein homolog of the presentinvention is from about 4 to about 6 amino acids in length. There is nolimit, other than a practical limit, on the maximal size of a nucleicacid molecule encoding a Der HMW-map protein of the present inventionbecause a nucleic acid molecule of the present invention can include aportion of a gene, an entire gene, or multiple genes. The preferred sizeof a protein encoded by a nucleic acid molecule of the present inventiondepends on whether a full-length, fusion, multivalent, or functionalportion of such a protein is desired. Preferably, the preferred size ofa protein encoded by a nucleic acid molecule of the present invention isa portion of the protein that induces an immune response which is about30 amino acids, more preferably about 35 amino acids and even morepreferably about 44 amino acids in length.

Stringent hybridization conditions are determined based on definedphysical properties of the gene to which the nucleic acid molecule isbeing hybridized, and can be defined mathematically. Stringenthybridization conditions are those experimental parameters that allow anindividual skilled in the art to identify significant similaritiesbetween heterologous nucleic acid molecules. These conditions are wellknown to those skilled in the art. See, for example, Sambrook, et al.,1989, Molecular Cloning. A Laboratory Manual, Cold Spring Harbor LabsPress, and Meinkoth, et al., 1984, Anal. Biochem. 138, 267-284, each ofwhich is incorporated by reference herein in its entirety. As explainedin detail in the cited references, the determination of hybridizationconditions involves the manipulation of a set of variables including theionic strength (M, in moles/liter), the hybridization temperature (°C.), the concentration of nucleic acid helix destabilizing agents (suchas formamide), the average length of the shortest hybrid duplex (n), andthe percent G+C composition of the fragment to which an unknown nucleicacid molecule is being hybridized. For nucleic acid molecules of atleast about 150 nucleotides, these variables are inserted into astandard mathematical formula to calculate the melting temperature, orT_(m), of a given nucleic acid molecule. As defined in the formulabelow, T_(m) is the temperature at which two complementary nucleic acidmolecule strands will disassociate, assuming 100% complementaritybetween the two strands:

T _(m)=81.5° C.+16.6 log M+0.41(%G+C)−500/n−0.61(%formamide).

For nucleic acid molecules smaller than about 50 nucleotides, hybridstability is defined by the dissociation temperature (T_(d)), which isdefined as the temperature at which 50% of the duplexes dissociate. Forthese smaller molecules, the stability at a standard ionic strength isdefined by the following equation:

T _(d)=4(G+C)+2(A+T).

A temperature of 5° C. below T_(d) is used to detect hybridizationbetween perfectly matched molecules.

Also well known to those skilled in the art is how base-pair mismatch,i.e. differences between two nucleic acid molecules being compared,including non-complementarity of bases at a given location, and gaps dueto insertion or deletion of one or more bases at a given location oneither of the nucleic acid molecules being compared, will affect T_(m)or T_(d) for nucleic acid molecules of different sizes. For example,T_(m) decreases about 1° C. for each 1% of mismatched base-pairs forhybrids greater than about 150 bp, and T_(d) decreases about 5° C. foreach mismatched base-pair for hybrids below about 50 bp. Conditions forhybrids between about 50 and about 150 base-pairs can be determinedempirically and without undue experimentation using standard laboratoryprocedures well known to those skilled in the art. These simpleprocedures allow one skilled in the art to set the hybridizationconditions (by altering, for example, the salt concentration, theformamide concentration or the temperature) so that only nucleic acidhybrids with less than a specified % base-pair mismatch will hybridize.Stringent hybridization conditions are commonly understood by thoseskilled in the art to be those experimental conditions that will allowhybridization between molecules having about 30% or less base-pairmismatch (i.e., about 70% or greater identity). Because one skilled inthe art can easily determine whether a given nucleic acid molecule to betested is less than or greater than about 50 nucleotides, and cantherefore choose the appropriate formula for determining hybridizationconditions, he or she can determine whether the nucleic acid moleculewill hybridize with a given gene under stringent hybridizationconditions and similarly whether the nucleic acid molecule willhybridize under conditions designed to allow a desired amount of basepair mismatch.

Hybridization reactions are often carried out by attaching the nucleicacid molecule to be hybridized to a solid support such as a membrane,and then hybridizing with a labeled nucleic acid molecule, typicallyreferred to as a probe, suspended in a hybridization solution. Examplesof common hybridization reaction techniques include, but are not limitedto, the well-known Southern and northern blotting procedures. Typically,the actual hybridization reaction is done under non-stringentconditions, i.e., at a lower temperature and/or a higher saltconcentration, and then high stringency is achieved by washing themembrane in a solution with a higher temperature and/or lower saltconcentration in order to achieve the desired stringency.

For example, if the skilled artisan wished to identify a nucleic acidmolecule that hybridizes under stringent hybridization conditions with aDermatophagoides farinae and/or Dermatophagoides pteronyssius nucleicacid molecule of about 150 bp in length, the following conditions couldpreferably be used. The average G+C content of Dermatophagoides farinaeand Dermatophagoides pteronyssius DNA is about 39%. The unknown nucleicacid molecules would be attached to a support membrane, and the 150 bpprobe would be labeled, e.g. with a radioactive tag. The hybridizationreaction could be carried out in a solution comprising 2×SSC and 0%formamide, at a temperature of about 37° C. (low stringency conditions).Solutions of differing concentrations of SSC can be made by one of skillin the art by diluting a stock solution of 20×SSC (175.3 gram NaCl andabout 88.2 gram sodium citrate in 1 liter of water, pH 7) to obtain thedesired concentration of SSC. In order to achieve high stringencyhybridization, the skilled artisan would calculate the washingconditions required to allow up to 30% base-pair mismatch. For example,in a wash solution comprising 1×SSC and 0% formamide, the T_(m) ofperfect hybrids would be about 80° C.:

81.5° C.+16.6 log(0.15M)+(0.41×39)−(500/150)−(0.61×0)=80.4° C.

Thus, to achieve hybridization with nucleic acid molecules having about30% base-pair mismatch, hybridization washes would be carried out at atemperature of about 50° C. It is thus within the skill of one in theart to calculate additional hybridization temperatures based on thedesired percentage base-pair mismatch, formulae and G/C contentdisclosed herein. For example, it is appreciated by one skilled in theart that as the nucleic acid molecule to be tested for hybridizationagainst nucleic acid molecules of the present invention having sequencesspecified herein becomes longer than 150 nucleotides, the T_(m) for ahybridization reaction allowing up to 30% base-pair mismatch will notvary significantly from 50° C.

Furthermore, it is known in the art that there are commerciallyavailable computer programs for determining the degree of similaritybetween two nucleic acid sequences. These computer programs includevarious known methods to determine the percentage identity and thenumber and length of gaps between hybrid nucleic acid molecules.Preferred methods to determine the percent identity among amino acidsequences and also among nucleic acid sequences include analysis usingone or more of the commercially available computer programs designed tocompare and analyze nucleic acid or amino acid sequences. These computerprograms include, but are not limited to, GCG™ (available from GeneticsComputer Group, Madison, Wis.), DNAsis™ (available from HitachiSoftware, San Bruno, Calif.) and MacVector™ (available from the EastmanKodak Company, New Haven, Conn.). A preferred method to determinepercent identity among amino acid sequences and also among nucleic acidsequences includes using the Compare function by maximum matching withinthe program DNAsis Version 2.1 using default parameters.

One embodiment of the present invention includes Der HMW-map proteins.In one embodiment, Der HMW-map proteins of the present invention includeproteins that, when submitted to reducing 12% Tris glycine SDS-PAGE,migrate as bands at a molecular weight of from about 98 kD to about 109kD, as shown in FIG. 1. The bands in FIG. 1 are obtained when proteinsare collected from Dermataphagoides farinae mites using the methoddescribed in detail in Example 1. Preferably, Der HMW-map proteins ofthe present invention includes proteins having a molecular weightranging from about 90 kD to about 120 kD, and more preferably from about98 kD to about 109 kD. Preferred Der HMW-map proteins of the presentinvention include mapA and mapB, the identification of which isdescribed in the Examples section.

In another embodiment, Der HMW-map proteins of the present inventioninclude proteins that, when submitted to reducing 14% Tris glycineSDS-PAGE, migrate as a band at a molecular weight of about 60 kD, asshown in FIG. 2. The band in FIG. 2 is obtained when proteins arecollected from Dermataphagoides farinae mites using the method describedin detail in Example 9. Preferably, Der HMW-map proteins of the presentinvention includes proteins having a molecular weight of about 60 kD.Preferred Der HMW-map proteins of the present invention include mapD,the identification of which is described in the Examples section.

In another embodiment, a preferred Der HMW-map protein includes aprotein encoded by a nucleic acid molecule which is at least about 50nucleotides, or about 150 nucleotides, and which hybridizes underconditions which preferably allow about 40% or less base pair mismatch,more preferably under conditions which allow about 35% or less base pairmismatch, more preferably under conditions which allow about 30% or lessbase pair mismatch, more preferably under conditions which allow about25% or less base pair mismatch, more preferably under conditions whichallow about 20% or less base pair mismatch, more preferably underconditions which allow about 15% or less base pair mismatch, morepreferably under conditions which allow about 10% or less base pairmismatch and even more preferably under conditions which allow about 5%or less base pair mismatch with a nucleic acid molecule selected fromthe group consisting of SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ IDNO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45 and a nucleic acidsequence encoding a protein comprising the amino acid sequence SEQ IDNO:33 the complement thereof

Another embodiment of the present invention includes a Der HMW-mapprotein encoded by a nucleic acid molecule selected from the groupconsisting of: a nucleic acid molecule comprising a t least about 150nucleotides, wherein said nucleic acid molecule comprising at leastabout 150 nucleotides hybridizes, in a solution comprising 1×SSC and 0%formamide, at a temperature of about 50° C., to a nucleic acid sequenceselected from the group consisting of SEQ ID NO:16, SEQ ID NO:19, SEQ IDNO:22, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, and acomplement of a nucleic acid sequence encoding a protein comprising theamino acid sequence SEQ ID NO.33; and a nucleic acid molecule comprisinga fragment of any of said nucleic acid molecules comprising at leastabout 15 nucleotides.

Yet another preferred Der HMW-map protein of the present inventionincludes a protein encoded by a nucleic acid molecule which ispreferably at least about 60% identical, more preferably at least about65% identical, more preferably at least about 70% identical, morepreferably at least about 75% identical, more preferably at least about80% identical, more preferably at least about 85% identical, morepreferably at least about 90% identical and even more preferably atleast about 95% identical to a nucleic acid molecule having the nucleicacid sequence SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:34,SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, and/or a complement of anucleic acid sequence encoding a protein comprising the amino acidsequence SEQ ID NO:33; also preferred are fragments of such proteins.Percent identity as used herein is determined using the Compare functionby maximum matching within the program DNAsis Version 2.1 using defaultparameters.

Additional preferred Der HMW-map proteins of the present inventioninclude proteins having the amino acid sequence SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, andproteins comprising homologs of a protein having the amino acid sequenceSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21,SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41,SEQ ID NO:44 in which such a homolog comprises at least one epitope thatelicits an immune response against a protein having an amino acidsequence SEQ ID NO 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:18,SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:30,SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:38,SEQ ID NO:41, SEQ ID NO:44 Likewise, also preferred are proteins encodedby nucleic acid molecules encoded by nucleic acid molecules havingnucleic acid sequence SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ IDNO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43 and/or a nucleic acidsequence encoding a protein comprising the amino acid sequence SEQ IDNO:33, or by homologs thereof.

A preferred isolated protein of the present invention is a proteinencoded by at least one of the following nucleic acid molecules:nDerf98₁₇₅₂, nDerf98₁₆₆₅, nDerf98₁₆₀₈, nDerp98₁₆₂₁, nDerp98₁₅₂₇,nDerp98₁₄₇₀, nDerf60₅₁₀, or allelic variants of any of these nucleicacid molecules. Another preferred isolated protein is encoded by anucleic acid molecule having nucleic acid sequence SEQ ID NO:14, SEQ IDNO:17, SEQ ID NO:20, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ IDNO:43; or a protein encoded by an allelic variant of any of these listednucleic acid molecule.

Translation of SEQ ID NO:14, the coding strand of nDerf98₁₇₅₂, yields aprotein of about 555 amino acids, denoted herein as PDerf98₅₅₅, theamino acid sequence of which is presented in SEQ ID NO:15, assuming afirst in-frame codon extending from nucleotide 1 to nucleotide 3 of SEQID NO:14. The complementary strand of SEQ ID NO:14 is presented hereinas SEQ ID NO:16. The amino acid sequence of PDerf98₅₅₅ is encoded by thenucleic acid molecule nDerf98₁₆₆₅, having a coding strand denoted SEQ IDNO:17 and a complementary strand denoted SEQ ID NO:19. Analysis of SEQID NO:15 suggests the presence of a signal peptide spanning from aboutamino acid 1 through about amino acid 19. The proposed mature protein,denoted herein as PDerf98₅₃₆, contains about 536 amino acids, thesequence of which is represented herein as SEQ ID NO:21, and is encodedby a nucleic acid molecule referred to herein as nDerf98₁₆₀₈,represented by SEQ ID NO:20, the coding strand, and SEQ ID NO:22, thecomplementary strand.

Translation of SEQ ID NO:34, the coding strand of nDerp98₁₆₂₁, yields aprotein of about 509 amino acids, denoted herein as PDerp98₅₀₉, theamino acid sequence of which is presented in SEQ ID NO:35, assuming afirst in-frame codon extending from nucleotide 14 to nucleotide 16 ofSEQ ID NO:34. The complementary strand of SEQ ID NO:34 is presentedherein as SEQ ID NO:36. The amino acid sequence of PDerpf98₅₀₉ isencoded by the nucleic acid molecule nDerp98₁₅₂₇, having a coding stranddenoted SEQ ID NO:37 and a complementary strand denoted SEQ ID NO:39.Analysis of SEQ ID NO:35 suggests the presence of a signal peptidespanning from about amino acid 1 through about amino acid 19. Theproposed mature protein, denoted herein as PDerp98₁₄₉₀, contains about490 amino acids, the sequence of which is represented herein as SEQ IDNO:41, and is encoded by a nucleic acid molecule referred to herein asnDerp98₁₄₇₀, represented by SEQ ID NO:40, the coding strand, and SEQ IDNO:42, the complementary strand.

Translation of SEQ ID NO:43, the coding strand of nDerf60₅₁₀, a nucleicacid molecule encoding a portion of the D. farinae 60-kD antigen proteinyields a protein of about 170 amino acids, denoted herein as PDerf60₁₇₀,the amino acid sequence of which is presented as SEQ ID NO:44, assuminga first in-frame codon extending from nucleotide 1 to nucleotide 3 ofSEQ ID NO:43. The complementary sequence to SEQ ID NO:43 is presentedherein as SEQ ID NO:45.

Preferred Der HMW-map proteins of the present invention include proteinsthat are at least about 45%, preferably at least about 50%, morepreferably at least about 55%, even more preferably at least about 60%,even more preferably at least about 65%, even more preferably at leastabout 70%, even more preferably at least about 75%, even more preferablyat least about 80%, even more preferably at least about 85%, even morepreferably at least about 90%, and even more preferably about 95%identical to PDerf98₅₅₅. More preferred is a Der HMW-map proteincomprising PDerf98₅₅₅, PDerf98₅₃₆, PDerp98₅₀₉, PDerp98₄₉₀, and/orPDerf60₁₇₀; and proteins encoded by allelic variants of nucleic acidmolecules encoding proteins PDerf98₅₅₅, PDerf98₅₃₆, PDerp98₅₀₉,PDerp98₄₉₀, and/or PDerf60₁₇₀.

Other preferred Der HMW-map proteins of the present invention includeproteins having amino acid sequences that are at least about 45%,preferably at least about 50%, more preferably at least about 55%, evenmore preferably at least about 60%, even more preferably at least about65%, even more preferably at least about 70%, even more preferably atleast about 75%, even more preferably at least about 80%, even morepreferably at least about 85%, even more preferably at least about 90%,and even more preferably about 95% identical to amino acid sequence SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21,SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41,and/or SEQ ID NO:44. More preferred are Der HMW-map proteins comprisingamino acid sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:18, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:38, SEQ ID NO:41, and/or SEQ ID NO:44; and Der HMW-map proteinsencoded by allelic variants of nucleic acid molecules encoding DerHMW-map proteins having amino acid sequences SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, and/or SEQ ID NO:44.

In one embodiment of the present invention, Der HMW-map proteinscomprise amino acid sequence SEQ ID NO:15, SEQ ID NO:35, and/or SEQ IDNO:44 (including, but not limited to, the proteins consisting of aminoacid sequence SEQ ID NO:15, SEQ ID NO:35, and/or SEQ ID NO:44, fragmentsthereof, fusion proteins and multivalent proteins), and proteins encodedby allelic variants of nucleic acid molecules encoding proteins havingamino acid sequence SEQ ID NO:15, SEQ ID NO:35, and/or SEQ ID NO:44.

In one embodiment, a preferred Der HMW-map protein comprises an aminoacid sequence of at least about 35 amino acids in length, preferably atleast about 50 amino acids in length, more preferably at least about 100amino acids in length, more preferably at least about 200 amino acids inlength, even more preferably at least about 250 amino acids in length.Within this embodiment, a preferred Der HMW-map protein of the presentinvention has an amino acid sequence comprising at least a-portion ofSEQ ID NO:15. In another embodiment, a preferred Der HMW-map proteincomprises a full-length protein, i.e., a protein encoded by afull-length coding region.

Additional preferred Der HMW-map proteins of the present inventioninclude proteins encoded by nucleic acid molecules comprising at least aportion of nDerfD98₁₇₅₂, nDerf98₁₆₆₅, nDerf98₁₆₀₈, nDerp98₁₆₂₁,nDerp98₁₅₂₇, nDerp98₁₄₇₀, and nDerf60₅₁₀, as well as Der HMW-mapproteins encoded by allelic variants of such nucleic acid molecules.

Also preferred are Der HMW-map proteins encoded by nucleic acidmolecules having nucleic acid sequences comprising at least a portion ofSEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:34, SEQ ID NO:37,SEQ ID NO:40 SEQ ID NO:43 and/or a nucleic acid sequence encoding aprotein comprising the amino acid sequence SEQ ID NO:33, as well asallelic variants of these nucleic acid molecules.

In another embodiment, a preferred Der HMW-map protein of the presentinvention is encoded by a nucleic acid molecule comprising at leastabout 12 nucleotides, preferably at least about 16 nucleotides, morepreferably at least about 18 nucleotides, more preferably at least about20 nucleotides, more preferably at least about 25 nucleotides, morepreferably at least about 50 nucleotides, more preferably at least about100 nucleotides, more preferably at least about 350 nucleotides, morepreferably at least about 450 nucleotides, more preferably at leastabout 500 nucleotides, and even more preferably at least about 800nucleotides. Within this embodiment is a Der HMW-map protein encoded byat least a portion nDerf98₁₇₅₂, nDerp98₁₆₂₁, and/or nDerf60₅₁₀ or by anallelic variant of these nucleic acid molecules. In yet anotherembodiment, a preferred Der HMW-map protein of the present invention isencoded by a nucleic acid molecule comprising an apparently full-lengthDer HMW-map coding region, i.e., a nucleic acid molecule encoding anapparently full-length Der HMW-map protein.

One embodiment of a Der HMW-map protein of the present invention is afusion protein that includes a Der HMW-map protein-containing domainattached to one or more fusion segments. Suitable fusion segments foruse with the present invention include, but are not limited to, segmentsthat can: enhance a protein's stability; act as an immunopotentiator toenhance an immune response against a Der HMW-map protein, reduce an IgEresponse against a Der HMW-map protein; and/or assist purification of aDer HMW-map protein (e.g., by affinity chromatography). A suitablefusion segment can be a domain of any size that has the desired function(e.g., imparts increased stability, imparts increased immunogenicity toa protein, reduces an IgE response, and/or simplifies purification of aprotein). Fusion segments can be joined to amino and/or carboxyl terminiof the Der HMW-map protein-containing domain of the protein and can besusceptible to cleavage in order to enable straight-forward recovery ofa Der HMW-map protein. Fusion proteins are preferably produced byculturing a recombinant cell transformed with a fusion nucleic acidmolecule that encodes a protein including the fusion segment attached toeither the carboxyl and/or amino terminal end of a Der HMW-mapprotein-containing domain. Preferred fusion segments include a metalbinding domain (e.g., a poly-histidine segment); an immunoglobulinbinding domain (e.g., Protein A; Protein G; T cell; B cell; Fc receptoror complement protein antibody-binding domains); a sugar binding domain(e.g., a maltose binding domain); a “tag” domain (e.g., at least aportion of—galactosidase, a strep tag peptide, other domains that can bepurified using compounds that bind to the domain, such as monoclonalantibodies); and/or a linker and enzyme domain (e.g., alkalinephosphatase domain connected to a Der HMW-map protein by a linker). Morepreferred fusion segments include metal binding domains, such as apoly-histidine segment; a maltose binding domain; a strep tag peptide,such as that available from Biometra in Tampa, Fla.; and a phage T7 S10peptide.

In another embodiment, a Der HMW-map protein of the present inventionalso includes at least one additional protein segment that is capable ofdesensitizing an animal from one or more allergens. Such a multivalentdesensitizing protein can be produced by culturing a cell transformedwith a nucleic acid molecule comprising two or more nucleic acid domainsjoined together in such a manner that the resulting nucleic acidmolecule is expressed as a multivalent desensitizing compound containingat least two desensitizing compounds capable of desensitizing an animalfrom allergens.

Examples of multivalent desensitizing compounds include, but are notlimited to, a Der HMW-map protein of the present invention attached toone or more compounds that desensitize against allergies caused by oneor more allergens, such as a plant allergen, an animal allergen, aparasite allergen or an ectoparasite allergen, including, but notlimited to: pant allergens from grass, Meadow Fescue, Curly Dock,plantain, Mexican Firebush, Lamb's Quarters, pigweed, ragweed, sage,elm, cocklebur, Box Elder, walnut, cottonwood, ash, birch, cedar, oak,mulberry, cockroach, Dermatophagoides, Alternaria, Aspergillus,Cladosporium, Fusarium, Helminthosporium, Mucor, Penicillium,Pullularia, Rhizopus and/or Tricophyton; parasite allergens fromhelminths; or ectoparasite allergens from arachnids, insects andleeches, including fleas, ticks, flies, mosquitos, sand flies, blackflies, horse flies, horn flies, deer flies, tsetse flies, stable flies,myiasis-causing flies and biting gnats, ants, spiders, lice; mites andtrue bugs.

The present invention also includes mimetopes of a Der HMW-map proteinof the present invention. As used herein, a mimetope of a Der HMW-mapprotein of the present invention refers to any compound that is able tomimic the activity of such a Der HMW-map protein (e.g., ability to bindto induce an immune response against Der HMW-map protein), often becausethe mimetope has a structure that mimics the Der HMW-map protein. It isto be noted, however, that the mimetope need not have a structuresimilar to a Der HMW-map protein as long as the mimetope functionallymimics the protein. Mimetopes can be, but are not limited to: peptidesthat have been modified to decrease their susceptibility to degradation;anti-idiotypic and/or catalytic antibodies, or fragments thereof;non-proteinaceous immunogenic portions of an isolated protein (e.g.,carbohydrate structures); synthetic or natural organic or inorganicmolecules, including nucleic acids; and/or any other peptidomimeticcompounds. Mimetopes of the present invention can be designed usingcomputer-generated structures of Der HMW-map protein of the presentinvention. Mimetopes can also be obtained by generating random samplesof molecules, such as oligonucleotides, peptides or other organicmolecules, and screening such samples by affinity chromatographytechniques using the corresponding binding partner, (e.g., an anti-DerHMW-map protein antibody). A mimetope can also be obtained by, forexample, rational drug design. In a rational drug design procedure, thethree-dimensional structure of a compound of the present invention canbe analyzed by, for example, nuclear magnetic resonance (NMR) or x-raycrystallography. The three-dimensional structure can then be used topredict structures of potential mimetopes by, for example, computermodeling. The predicted mimetope structures can then be produced by, forexample, chemical synthesis, recombinant DNA technology, or by isolatinga mimetope from a natural source. Specific examples of Der HMW-mapprotein mimetopes include anti-idiotypic antibodies, oligonucleotidesproduced using Selex™ technology, peptides identified by randomscreening of peptide libraries and proteins identified by phage displaytechnology. A preferred mimetope is a peptidomimetic compound that isstructurally and/or functionally similar to a Der HMW-map protein of thepresent invention, particularly to an epitope of Der HMW-map proteinthat induces an immune response.

The present invention also includes muteins of a Der HMW-map protein ofthe present invention. As used herein, a mutein refers to a particularhomolog of a Der HMW-map protein in which desired amino acid residueshave been substituted or removed. Preferred muteins of the presentinvention include Der HMW-map protein homologs in which amino acidresidues have been changed to reduce an anaphylactic reaction by ananimal when the mutein is administered to the animal in therapeuticdoses. More preferred muteins of the present invention include DerHMW-map protein homologs in which one or more cysteine residues of a DerHMW-map protein have been replaced or removed. Methods to producemuteins are known to those of skill in the art and are disclosed herein.Preferably, a mutein is produced using recombinant techniques.

Another embodiment of the present invention is an isolated nucleic acidmolecule comprising a Der HMW-map nucleic acid molecule. The identifyingcharacteristics of such nucleic acid molecules are heretofore described.A nucleic acid molecule of the present invention can include an isolatednatural Der HMW-map gene or a homolog thereof, the latter of which isdescribed in more detail below. A nucleic acid molecule of the presentinvention can include one or more regulatory regions, full-length orpartial coding regions, or combinations thereof. The minimal size of anucleic acid molecule of the present invention is a size sufficient toallow the formation of a stable hybrid (i.e., hybridization understringent hybridization conditions) with the complementary sequence ofanother nucleic acid molecule.

In accordance with the present invention, an isolated nucleic acidmolecule is a nucleic acid molecule that has been removed from itsnatural milieu (i.e., that has been subjected to human manipulation) andcan include DNA, RNA, or derivatives of either DNA or RNA. As such,“isolated” does not reflect the extent to which the nucleic acidmolecule has been purified. An isolated Der HMW-map nucleic acidmolecule of the present invention, or a homolog thereof, can be isolatedfrom its natural source or produced using recombinant DNA technology(e.g., polymerase chain reaction (PCR) amplification or cloning) orchemical synthesis. Isolated Der HMW-map nucleic acid molecules, andhomologs thereof, can include, for example, natural allelic variants andnucleic acid molecules modified by nucleotide insertions, deletions,substitutions, and/or inversions in a manner such that the modificationsdo not substantially interfere with the nucleic acid molecule's abilityto encode a Der HMW-map protein of the present invention.

A Der HMW-map nucleic acid molecule homolog can be produced using anumber of methods known to those skilled in the art, see, for example,Sambrook et al., 1989, Molecular Cloning. A Laboratory Manual, ColdSpring Harbor Labs Press; Sambrook et al., ibid., is incorporated byreference herein in its entirety. For example, nucleic acid moleculescan be modified using a variety of techniques including, but not limitedto, classic mutagenesis and recombinant DNA techniques such assite-directed mutagenesis, chemical treatment, restriction enzymecleavage, ligation of nucleic acid fragments, PCR amplification,synthesis of oligonucleotide mixtures and ligation of mixture groups to“build” a mixture of nucleic acid molecules, and combinations thereof.Nucleic acid molecule homologs can be selected by hybridization with aDer HMW-map nucleic acid molecule or by screening the function of aprotein encoded by the nucleic acid molecule (e.g., ability to elicit animmune response against at least one epitope of a Der HMW-map protein orto effect Der HMW-map activity).

Allelic variants typically encode proteins having similar activity tothat of the protein encoded by the gene to which they are beingcompared. Allelic variants can also comprise alterations in the 5′ or 3′untranslated regions of the gene (e.g., in regulatory control regions).Allelic variants are well known to those skilled in the art and would beexpected to be found within a given dust mite since the genome isdiploid and/or among a group of two or more dust mites. The presentinvention also includes variants due to laboratory manipulation, suchas, but not limited to, variants produced during polymerase chainreaction amplification.

An isolated nucleic acid molecule of the present invention can include anucleic acid sequence that encodes at least one Der HMW-map protein ofthe present invention, examples of such proteins being disclosed herein.Although the phrase “nucleic acid molecule” primarily refers to thephysical nucleic acid molecule and the phrase “nucleic acid sequence”primarily refers to the sequence of nucleotides on the nucleic acidmolecule, the two phrases can be used interchangeably, especially withrespect to a nucleic acid molecule, or a nucleic acid sequence, beingcapable of encoding a Der HMW-map protein.

A preferred nucleic acid molecule of the present invention, whenadministered to an animal, is capable of desensitizing that animal fromallergic reactions caused by a Der HMW-map allergen. As will bedisclosed in more detail below, such a nucleic acid molecule can be, orencode, an antisense RNA, a molecule capable of triple helix formation,a ribozyme, or other nucleic acid-based drug compound. In additionalembodiments, a nucleic acid molecule of the present invention can encodea desensitizing protein (e.g., a Der HMW-map protein of the presentinvention), the nucleic acid molecule being delivered to the animal, forexample, by direct injection (i.e, as a DNA reagent) or in a vehiclesuch as a recombinant virus reagent or a recombinant cell reagent.

One embodiment of the present invention is an isolated nucleic acidmolecule that hybridizes under stringent hybridization conditions with aDer HMW-map gene. Stringent hybridization conditions refer to standardhybridization conditions described herein. A preferred nucleic acidmolecule of the present invention includes an isolated nucleic acidmolecule that hybridizes under stringent hybridization conditions with agene encoding a protein comprising an amino acid sequence including SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21,SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41,and/or SEQ ID NO:44. A more preferred nucleic acid molecule of thepresent invention includes an isolated nucleic acid molecule thathybridizes under stringent hybridization conditions with the complementof a nucleic acid sequence that encodes a protein comprising an aminoacid sequence including SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15,SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35,SEQ ID NO:38, SEQ ID NO:41, and/or SEQ ID NO:44.

A more preferred nucleic acid molecule of the present invention includesan isolated nucleic acid molecule selected from the group consisting of:a nucleic acid molecule comprising at least about 150 nucleotides,wherein said nucleic acid molecule comprising at least about 150nucleotides hybridizes, in a solution comprising 1×SSC and 0% formamide,at a temperature of about 50° C., to a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17,SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:34, SEQ ID NO:36,SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43,SEQ ID NO:45 and/or a nucleic acid sequence encoding a proteincomprising the amino acid sequence SEQ ID NO:33 and a complementthereof.

The present invention also includes fragments of any nucleic acidmolecule disclosed herein. According to the present invention, afragment can include any nucleic acid molecule or nucleic acid sequence,the size of which can range between a length that is smaller than asequence identified by a SEQ ID NO of the present invention and theminimum size of an oligonucleotide as defined herein. For example, thesize of a fragment of the present invention can be any size that is lessthan about 1752 nucleotides and greater than 11 nucleotides in length.

In one embodiment of the present invention, a preferred Der HMW-mapnucleic acid molecule includes an isolated nucleic acid molecule whichis at least about 50 nucleotides, or at least about 150 nucleotides, andwhich hybridizes under conditions which preferably allow about 40% orless base pair mismatch, more preferably under conditions which allowabout 35% or less base pair mismatch, more preferably under conditionswhich allow about 30% or less base pair mismatch, more preferably underconditions which allow about 25% or less base pair mismatch, morepreferably under conditions which allow about 20% or less base pairmismatch, more preferably under conditions which allow about 15% or lessbase pair mismatch, more preferably under conditions which allow about10% or less base pair mismatch and even more preferably under conditionswhich allow about 5% or less base pair mismatch with a nucleic acidmolecule selected from the group consisting of SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:43, SEQ ID NO:45, and a nucleic acid sequence encoding a proteincomprising the amino acid sequence SEQ ID NO:33 and a complementthereof.

Another embodiment of the present invention includes a nucleic acidmolecule comprising at least about 150 base-pairs, wherein the nucleicacid molecule hybridizes, in a solution comprising 1×SSC and 0%formamide, at a temperature of about 50° C., to a nucleic acid sequenceselected from the group consisting of SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:43, SEQ ID NO:45, and/or a nucleic acid sequence encoding a proteincomprising the amino acid sequence SEQ ID NO:33 and a complementthereof. Additional preferred nucleic acid molecules of the presentinvention include fragments of an isolated nucleic acid moleculecomprising at least about 150 base-pairs, wherein said nucleic acidmolecule hybridizes, in a solution comprising 1×SSC and 0% formamide, ata temperature of about 50° C., to a nucleic acid sequence selected fromthe group consisting of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:34, SEQ ID NO:36, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ IDNO:45 and a nucleic acid sequence encoding a protein comprising theamino acid sequence SEQ ID NO:33 and complement thereof.

Additional preferred Der HMW-map nucleic acid molecules of the presentinvention include an isolated nucleic acid molecule which is at leastabout 50 nucleotides, or at least about 150 nucleotides, comprising anucleic acid sequence that is preferably at leas t about 60% identical,more preferably at least about 65% identical, more preferably at leastabout 70% identical, more preferably at least about 75% identical, morepreferably at least about 80% identical, more preferably at least about85% identical, more preferably at least about 90% identical and evenmore preferably at least about 95% identical to a nucleic acid sequenceselected from the group consisting of SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:43, SEQ ID NO:45, and a nucleic acid sequence encoding a proteincomprising the amino acid sequence SEQ ID NO:33 and a complementthereof. Also preferred are fragments of any of such nucleic acidmolecules. Percent identity may be determined using the Compare functionby maximum matching within the program DNAsis Version 2.1 using defaultparameters.

One embodiment of the present invention is a nucleic acid moleculecomprising all or part of nucleic acid molecules nDerf98₁₇₅₂,nDerf98₁₆₆₅ and nDerf98₁₆₀₈, nDerp98₁₆₂₁, nDerp98₁₅₂₇, nDerp98₁₄₇₀,and/or nDerf60₅₁₀, or allelic variants of these nucleic acid molecules.Another preferred nucleic acid molecule of the present inventionincludes at least a portion of nucleic acid sequence SEQ ID NO:14, SEQID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ IDNO:42, SEQ ID NO:43, SEQ ID NO:45 and/or a nucleic acid sequenceencoding a protein comprising the amino acid sequence SEQ ID NO:33, aswell as allelic variants of nucleic acid molecules having these nucleicacid sequences and homologs of nucleic acid molecules having thesenucleic acid sequences; preferably such a homolog encodes or iscomplementary to a nucleic acid molecule that encodes at least oneepitope that elicits and an immune response against a protein having anamino acid sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:18, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:38, SEQ ID NO:41, SEQ ID NO:41, and/or SEQ ID NO:44. Such nucleicacid molecules can include nucleotides in addition to those included inthe SEQ ID NOs, such as, but not limited to, a full-length gene, afull-length coding region, a nucleic acid molecule encoding a fusionprotein, or a nucleic acid molecule encoding a multivalent protectivecompound.

In one embodiment, a Der HMW-map nucleic acid molecule of the presentinvention encodes a protein that is at least about 45%, preferably atleast about 50%, more preferably at least about 55%, even morepreferably at least about 60%, even more preferably at least about 65%,even more preferably at least about 70%, even more preferably at leastabout 75%, even more preferably at least about 80%, even more preferablyat least about 85%, even more preferably at least about 90%, and evenmore preferably about 95% identical to PDerf98₅₅₅, PDerp98₅₀₉, and/orPDerf60₁₇₀. Even more preferred is a nucleic acid molecule encodingPDerf98₅₅₅, PDerf98₅₃₆, PDerp98₅₀₉, PDerp98₄₉₀, and/or PDerf60₁₇₀,and/or an allelic variant of such nucleic acid molecules.

In another embodiment, a Der HMW-map nucleic acid molecule of thepresent invention encodes a protein having an amino acid sequence thatis at least about 45%, preferably at least about 50%, more preferably atleast about 55%, even more preferably at least about 60%, even morepreferably at least about 65%, even more preferably at least about 70%,even more preferably at least about 75%, even more preferably at leastabout 80%, even more preferably at least about 85%, even more preferablyat least about 90%, and even more preferably about 95% identical to SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:18 , SEQ ID NO:21,SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41,SEQ ID NO:41, and/or SEQ ID NO:44. The present invention also includes aDer HMW-map nucleic acid molecule encoding a protein having at least aportion of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:18,SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:30,SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:38,SEQ ID NO:41, SEQ ID NO:41, and/or SEQ ID NO:44, as well as allelicvariants of a Der HMW-map nucleic acid molecule encoding a proteinhaving these sequences, including nucleic acid molecules that have beenmodified to accommodate codon usage properties of the cells in whichsuch nucleic acid molecules are to be expressed.

In another embodiment, a preferred Der HMW-map nucleic acid moleculeencodes a Der HMW-map protein comprising at least about at least about35 amino acids in length, preferably at least about 50 amino acids inlength, more preferably at least about 100 amino acids in length, morepreferably at least about 200 amino acids in length, even morepreferably at least about 250 amino acids in length.

Knowing the nucleic acid sequences of certain Der HMW-map nucleic acidmolecules of the present invention allows one skilled in the art to, forexample, (a) make copies of those nucleic acid molecules, (b) obtainnucleic acid molecules including at least a portion of such nucleic acidmolecules (e.g., nucleic acid molecules including full-length genes,full-length coding regions, regulatory control sequences, truncatedcoding regions), and (c) obtain other Der HMW-map nucleic acidmolecules. Such nucleic acid molecules can be obtained in a variety ofways including screening appropriate expression libraries withantibodies of the present invention; traditional cloning techniquesusing oligonucleotide probes of the present invention to screenappropriate libraries; and PCR amplification of appropriate libraries orDNA using oligonucleotide primers of the present invention. A preferredlibrary to screen or from which to amplify nucleic acid moleculesincludes a Dermatophagoides farinae and/or Dermatophagoides pteronyssiuslibrary, such as the libraries disclosed herein in the Examples.Techniques to clone and amplify genes are disclosed, for example, inSambrook et al., ibid.

The present invention also includes nucleic acid molecules that areoligonucleotides capable of hybridizing, under stringent hybridizationconditions, with complementary regions of other, preferably longer,nucleic acid molecules of the present invention such as those comprisingDer HMW-map nucleic acid molecules or other Der HMW-map nucleic acidmolecules. Oligonucleotides of the present invention can be RNA, DNA, orderivatives of either. The minimum size of such oligonucleotides is thesize required for formation of a stable hybrid between anoligonucleotide and a complementary sequence on a nucleic acid moleculeof the present invention. A preferred oligonucleotide of the presentinvention has a maximum size of preferably about 200 nucleotides, morepreferably about 150 nucleotides and even more preferably about 100nucleotides. The present invention includes oligonucleotides that can beused as, for example, probes to identify nucleic acid molecules.

One embodiment of the present invention includes a recombinant vector,which includes at least one isolated nucleic acid molecule of thepresent invention, inserted into any vector capable of delivering thenucleic acid molecule into a host cell. Such a vector containsheterologous nucleic acid sequences, that is nucleic acid sequences thatare not naturally found adjacent to nucleic acid molecules of thepresent invention and that preferably are derived from a species otherthan the species from which the nucleic acid molecule(s) are derived.The vector can be either RNA or DNA, either prokaryotic or eukaryotic,and typically is a virus or a plasmid. Recombinant vectors can be usedin the cloning, sequencing, and/or otherwise manipulation of Der HMW-mapnucleic acid molecules of the present invention.

One type of recombinant vector, referred to here in as a recombinantmolecule, comprises a nucleic acid molecule of the present inventionoperatively linked to an express ion vector. The phrase operativelylinked refers to insertion of a nucleic acid molecule into an expressionvector in a manner such that the molecule is able to be expressed whentransformed into a host cell. As used herein, an expression vector is aDNA or RNA vector that is capable of transforming a host cell and ofeffecting expression of a specified nucleic acid molecule. Preferably,the expression vector is also capable of replicating within the hostcell. Expression vectors can be either prokaryotic or eukaryotic, andare typically viruses or plasmids. Expression vectors of the presentinvention include any vectors that function (i.e., direct geneexpression) in recombinant cells of the present invention, including inbacterial, fungal, endoparasite, insect, other animal, and plant cells.Preferred expression vectors of the present invention can direct geneexpression in bacterial, yeast, insect and mammalian cells and morepreferably in the cell types disclosed herein.

In particular, expression vectors of the present invention containregulatory sequences such as transcription control sequences,translation control sequences, origins of replication, and otherregulatory sequences that are compatible with the recombinant cell andthat control the expression of nucleic acid molecules of the presentinvention. In particular, recombinant molecules of the present inventioninclude transcription control sequences. Transcription control sequencesare sequences which control the initiation, elongation, and terminationof transcription. Particularly important transcription control sequencesare those which control transcription initiation, such as promoter,enhancer, operator and repressor sequences. Suitable transcriptioncontrol sequences include any transcription control sequence that canfunction in at least one of the recombinant cells of the presentinvention. A variety of such transcription control sequences are knownto those skilled in the art. Preferred transcription control sequencesinclude those which function in bacterial, yeast, insect and mammaliancells, such as, but not limited to, tac, lac, trp, trc, oxy-pro,omp/lpp, rrnB, bacteriophage lambda (such as lambda p_(L) and lambdap_(R) and fusions that include such promoters), bacteriophage T7, T7lac,bacteriophage T3, bacteriophage SP6, bacteriophage SP01,metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirussubgenomic promoters (such as Sindbis virus subgenomic promoters),antibiotic resistance gene, baculovirus, Heliothis zea insect virus,vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus,adenovirus, cytomegalovirus (such as intermediate early promoters),simian virus 40, retrovirus, actin, retroviral long terminal repeat,Rous sarcoma virus, heat shock, phosphate and nitrate transcriptioncontrol sequences as well as other sequences capable of controlling geneexpression in prokaryotic or eukaryotic cells. Additional suitabletranscription control sequences include tissue-specific promoters andenhancers as well as lymphokine-inducible promoters (e.g., promotersinducible by interferons or interleukins). Transcription controlsequences of the present invention can also include naturally occurringtranscription control sequences naturally associated with canines orfelines.

Suitable and preferred nucleic acid molecules to include in recombinantvectors of the present invention are as disclosed herein. Preferrednucleic acid molecules to include in recombinant vectors, andparticularly in recombinant molecules, include nDerf98₁₇₅₂, nDerf98₁₆₆₅nDerf98₁₆₀₈, nDerp98₁₆₂₁, nDerp98₁₅₂₇, nDerp98₁₄₇₀, and nDerf60₅₁₀.

Recombinant molecules of the present invention may also (a) containsecretory signals (i.e., signal segment nucleic acid sequences) toenable an expressed Der HMW-map protein of the present invention to besecreted from the cell that produces the protein and/or (b) containfusion sequences which lead to the expression of nucleic acid moleculesof the present invention as fusion proteins. Examples of suitable signalsegments include any signal segment capable of directing the secretionof a protein of the present invention. Preferred signal segmentsinclude, but are not limited to, tissue plasminogen activator (t-PA),interferon, interleukin, growth hormone, histocompatibility and viralenvelope glycoprotein signal segments, as well as natural signalsegments. Suitable fusion segments encoded by fusion segment nucleicacids are disclosed herein. In addition, a nucleic acid molecule of thepresent invention can be joined to a fusion segment that directs theencoded protein to the proteosome, such as a ubiquitin fusion segment.Recombinant molecules may also include intervening and/or untranslatedsequences surrounding and/or within the nucleic acid sequences ofnucleic acid molecules of the present invention.

Another embodiment of the present invention includes a recombinant cellcomprising a host cell transformed with one or more recombinantmolecules of the present invention. Transformation of a nucleic acidmolecule into a cell can be accomplished by any method by which anucleic acid molecule can be inserted into the cell. Transformationtechniques include, but are not limited to, transfection,electroporation, microinjection, lipofection, adsorption, and protoplastfusion. A recombinant cell may remain unicellular or may grow into atissue, organ or a multicellular organism. Transformed nucleic acidmolecules of the present invention can remain extrachromosomal or canintegrate into one or more sites within a chromosome of the transformed(i.e., recombinant) cell in such a manner that their ability to beexpressed is retained. Preferred nucleic acid molecules with which totransform a cell include Der HMW-map nucleic acid molecules disclosedherein. Particularly preferred nucleic acid molecules with which totransform a cell include nDerf98₁₇₅₂, nDerf98₁₆₆₅ nDerf98₁₆₀₈,nDerp98₁₆₂₁, nDerp98₁₅₂₇, nDerp98₁₄₇₀, and nDerf60₅₁₀.

Suitable host cells to transform include any cell that can betransformed with a nucleic acid molecule of the present invention. Hostcells can be either untransformed cells or cells that are alreadytransformed with at least one nucleic acid molecule (e.g., nucleic acidmolecules encoding one or more proteins of the present invention and/orother proteins useful in the production of multivalent vaccines). Hostcells of the present invention either can be endogenously (i.e.,naturally) capable of producing Der HMW-map proteins of the presentinvention or can be capable of producing such proteins after beingtransformed with at least one nucleic acid molecule of the presentinvention. Host cells of the present invention can be any cell capableof producing at least one protein of the present invention, and includebacterial, fungal (including yeast), other insect, other animal andplant cells. Preferred host cells include bacterial, mycobacterial,yeast, parasite, insect and mammalian cells. More preferred host cellsinclude Salmonella, Escherichia, Bacillus, Listeria, Saccharomyces,Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells,MDCK cells (normal dog kidney cell line for canine herpesviruscultivation), CRFK cells (normal cat kidney cell line for felineherpesvirus cultivation), CV-1 cells (African monkey kidney cell lineused, for example, to culture raccoon poxvirus), COS (e.g., COS-7)cells, and Vero cells. Particularly preferred host cells are Escherichiacoli, including E. coli K-12 derivatives; Salmonella typhi; Salmonellatyphimurium, including attenuated strains such as UK-1 _(X)3987 andSR-11 _(X)4072; Spodoptera frugiperda; Trichoplusia ni; BHK cells; MDCKcells; CRFK cells; CV-1 cells; COS cells; Vero cells; andnon-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246).Additional appropriate mammalian cell hosts include other kidney celllines, other fibroblast cell lines (e.g., human, murine or chickenembryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovarycells, mouse NIH/3T3 cells, LMTK³¹ cells and/or HeLa cells.

A recombinant cell is preferably produced by transforming a host cellwith one or more recombinant molecules, each comprising one or morenucleic acid molecules of the present invention operatively linked to anexpression vector containing one or more transcription controlsequences. The phrase operatively linked refers to insertion of anucleic acid molecule into an expression vector in a manner such thatthe molecule is able to be expressed when transformed into a host cell.

A recombinant molecule of the present invention is a molecule that caninclude at least one of any nucleic acid molecule heretofore describedoperatively linked to at least one of any transcription control sequencecapable of effectively regulating expression of the nucleic acidmolecule(s) in the cell to be transformed, examples of which aredisclosed herein.

A recombinant cell of the present invention includes any celltransformed with at least one of any Der HMW-map nucleic acid moleculeof the present invention. Suitable and preferred Der HMW-map nucleicacid molecules as well as suitable and preferred recombinant moleculeswith which to transform cells are disclosed herein.

Recombinant DNA technologies can be used to improve expression oftransformed nucleic acid molecules by manipulating, for example, thenumber of copies of the nucleic acid molecules within a host cell, theefficiency with which those nucleic acid molecules are transcribed, theefficiency with which the resultant transcripts are translated, and theefficiency of post-translational modifications. Recombinant techniquesuseful for increasing the expression of nucleic acid molecules of thepresent invention include, but are not limited to, operatively linkingnucleic acid molecules to high-copy number plasmids, integration of thenucleic acid molecules into one or more host cell chromosomes, additionof vector stability sequences to plasmids, substitutions ormodifications of transcription control signals (e.g., promoters,operators, enhancers), substitutions or modifications of translationalcontrol signals (e.g., ribosome binding sites, Shine-Dalgarnosequences), modification of nucleic acid molecules of the presentinvention to correspond to the codon usage of the host cell, deletion ofsequences that destabilize transcripts, and use of control signals thattemporally separate recombinant cell growth from recombinant enzymeproduction during fermentation. The activity of an expressed recombinantprotein of the present invention may be improved by fragmenting,modifying, or derivatizing nucleic acid molecules encoding such aprotein.

Isolated Der HMW-map proteins of the present invention can be producedin a variety of ways, including production and recovery of naturalproteins, production and recovery of recombinant proteins, and chemicalsynthesis of the proteins. In one embodiment, an isolated protein of thepresent invention is produced by culturing a cell capable of expressingthe protein under conditions effective to produce the protein, andrecovering the protein. A preferred cell to culture is a recombinantcell of the present invention. Effective culture conditions include, butare not limited to, effective media, bioreactor, temperature, pH andoxygen conditions that permit protein production. An effective mediumrefers to any medium in which a cell is cultured to produce a DerHMW-map protein of the present invention. Such a medium typicallycomprises an aqueous medium having assimilable carbon, nitrogen andphosphate sources, and appropriate salts, minerals, metals and othernutrients, such as vitamins. Cells of the present invention can becultured in conventional fermentation bioreactors, shake flasks, testtubes, microtiter dishes, and petri plates. Culturing can be carried outat a temperature, pH and oxygen content appropriate for a recombinantcell. Such culturing conditions are within the expertise of one ofordinary skill in the art.

Depending on the vector and host system used for production, resultantproteins of the present invention may either remain within therecombinant cell; be secreted into the fermentation medium; be secretedinto a space between two cellular membranes, such as the periplasmicspace in E. coli; or be retained on the outer surface of a cell or viralmembrane. The phrase “recovering the protein”, as well as similarphrases, refers to collecting the whole fermentation medium containingthe protein and need not imply additional steps of separation orpurification. Proteins of the present invention can be purified using avariety of standard protein purification techniques, such as, but notlimited to, affinity chromatography, ion exchange chromatography,filtration, electrophoresis, hydrophobic interaction chromatography, gelfiltration chromatography, reverse phase chromatography, concanavalin Achromatography, chromatofocusing and differential solubilization.Proteins of the present invention are preferably retrieved in“substantially pure” form. As used herein, “substantially pure” refersto a purity that allows for the effective use of the protein as atherapeutic composition or diagnostic. A therapeutic composition foranimals, for example, should exhibit no substantial toxicity andpreferably should be capable of desensitizing a treated animal.

The present invention also includes isolated (i.e., removed from theirnatural milieu) antibodies that selectively bind to a Der HMW-mapprotein of the present invention or a mimetope thereof (i.e., anti-DerHMW-map protein antibodies). As used herein, the term “selectively bindsto” a Der HMW-map protein refers to the ability of antibodies of thepresent invention to preferentially bind to specified proteins andmimetopes thereof of the present invention. Binding can be measuredusing a variety of methods standard in the art including enzymeimmunoassays (e.g., ELISA), immunoblot assays, etc.; see, for example,Sambrook et al., ibid. An anti-Der HMW-map protein antibody preferablyselectively binds to a portion of a Der HMW-map protein that induces animmune response in an animal.

Isolated antibodies of the present invention can include antibodies in abodily fluid (such as, but not limited to, serum), or antibodies thathave been purified to varying degrees. Antibodies of the presentinvention can be polyclonal or monoclonal. Functional equivalents ofsuch antibodies, such as antibody fragments and genetically-engineeredantibodies (including single chain antibodies or chimeric antibodiesthat can bind to more than one epitope) are also included in the presentinvention.

A preferred method to produce antibodies of the present inventionincludes (a) administering to an animal an effective amount of aprotein, peptide or mimetope thereof of the present invention to producethe antibodies and (b) recovering the antibodies. In another method,antibodies of the present invention are produced recombinantly usingtechniques as heretofore disclosed to produce Der HMW-map proteins ofthe present invention. Antibodies raised against defined proteins ormimetopes can be advantageous because such antibodies are notsubstantially contaminated with antibodies against other substances thatmight otherwise cause interference in a diagnostic assay or side effectsif used in a therapeutic composition.

Antibodies of the present invention have a variety of potential usesthat are within the scope of the present invention. For example, suchantibodies can be used (a) as tools to detect mite allergen, inparticular Der HMW-map protein; (b) as tools to screen expressionlibraries; and/or (c) to recover desired proteins of the presentinvention from a mixture of proteins and other contaminants. Antibodiesof the present invention can also be used, for example, to inhibitbinding of Der HMW-map protein to IgE that binds specifically to DerHMW-map protein, to prevent immunocomplex formation, thereby reducinghypersensitivity responses to mite allergens.

A Der HMW-map protein of the present invention can be included in achimeric molecule comprising at least a portion of a Der HMW-map proteinthat induces an immune response in an animal and a second molecule thatenables the chimeric molecule to be bound to a substrate in such amanner that the Der HMW-map protein portion can bind to IgE inessentially the same manner as a Der HMW-map protein that is not boundto a substrate. An example of a suitable second molecule includes aportion of an immunoglobulin molecule or another ligand that has asuitable binding partner that can be immobilized on a substrate, e.g.,biotin and avidin, or a metal-binding protein and a metal (e.g., His),or a sugar-binding protein and a sugar (e.g., maltose).

A Der HMW-map protein of the present invention can be contained in aformulation, herein referred to as a Der HMW-map protein formulation.For example, a Der HMW-map protein can be combined with a buffer inwhich the Der HMW-map protein is solubilized, and/or with a carrier.Suitable buffers and carriers are known to those skilled in the art.Examples of suitable buffers include any buffer in which a Der HMW-mapprotein can function to selectively bind to an antibody thatspecifically binds to Der HMW-map protein, such as, but not limited to,phosphate buffered saline, water, saline, phosphate buffer, bicarbonatebuffer, HEPES buffer (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonicacid buffered saline), TES buffer (Tris-EDTA buffered saline), Trisbuffer and TAE buffer (Tris-acetate-EDTA). Examples of carriers include,but are not limited to, polymeric matrices, toxoids, and serum albumins,such as bovine serum albumin. Carriers can be mixed with Der HMW-mapprotein or conjugated (i.e., attached) to Der HMW-map protein in such amanner as to not substantially interfere with the ability of the DerHMW-map protein to selectively bind to an antibody that specificallybinds to Der HMW-map protein.

A Der HMW-map protein of the present invention can be produced by a cellcomprising the Der HMW-map protein. A preferred Der HMW-mapprotein-bearing cell includes a recombinant cell comprising a nucleicacid molecule encoding a Der HMW-map protein of the present invention.

In addition, a Der HMW-map protein formulation of the present inventioncan include not only a Der HMW-map protein but also one or moreadditional antigens or antibodies useful in desensitizing an animalagainst allergy, or preventing or treating mite allergen pathogenesis.As used herein, an antigen refers to any molecule capable of beingselectively bound by an antibody. As used herein, an allergen refers toany antigen that is capable of stimulating production of antibodiesinvolved in an allergic response in an animal. As used herein, selectivebinding of a first molecule to a second molecule refers to the abilityof the first molecule to preferentially bind (e.g., having higheraffinity higher avidity) to the second molecule when compared to theability of a first molecule to bind to a third molecule. The firstmolecule need not necessarily be the natural ligand of the secondmolecule. Allergens of the present invention are preferably derived frommites, and mite-related allergens including, but not limited to, otherinsect allergens and plant allergens.

In accordance with the present invention, virtually any substance canact as an antigen and elicit an antibody response, i.e., can function asan epitope. For example, antibodies can be raised in response tocarbohydrate epitopes, including saccharides and/or polysaccharides thatare attached to a protein, a so-called glycosylated protein. However, asaccharide and/or polysaccharide may act as an antigen alone, without aprotein being present. The terminal sugar of a carbohydrate moiety, aswell as internal sugars can serve as an epitope. Polysaccharide may bepresent as a branched chain, in which case epitopes may comprise sugarsthat are not contiguous in sequence, but are adjacent spatially.Unusual, insect-specific sugars, not normally seen in mammalianproteins, may be present on glycoprotein derived from insect nucleicacid molecules, and these unusual sugars can comprise an epitoperecognized by a mammalian immune system.

One embodiment of the present invention is a reagent comprising anon-proteinaceous epitope that is capable of binding to IgE of an animalthat is allergic to mites, of desensitizing an animal against miteallergen, of stimulating a B lymphocyte response, and/or of stimulatinga T lymphocyte response. Such an epitope, referred to herein as a Der NPepitope, can exist as part of a Der HMW-map protein of the presentinvention or can be isolated therefrom. Such an epitope exists, forexample, on a protein contained in the D. farinae HMW-map compositionproduced in accordance with Example 1. A Der NP epitope of the presentinvention can be isolated from its natural source or producedsynthetically. Such an epitope can be, but need not be, joined to acarrier or other molecule. A Der NP epitope has at least one of thefollowing identifying characteristics: (a) the epitope is resistant toβ-elimination of peptides; (b) the epitope is resistant to Proteinase-Kdigestion; and (c) the epitope is reactive to a test designed to detectglycosylated proteins. A preferred Der NP epitope has all suchidentifying characteristics. A Der NP epitope can selectively bind toIgE of dogs or cats that are allergic to mites. While not being bound bytheory, it is believed that a Der NP epitope comprises a carbohydratemoiety that apparently does not include an N-linked glycan.Identification of the structural characteristics of such an epitope canbe determined by one skilled in the art. In one embodiment, there isprovided an isolated antibody that selectively binds to a Der NPepitope. The present invention also includes a derivative of a Der NPepitope, i.e., a compound that mimics the activity of such an epitope(e.g. is a Der NP epitope mimetope) and is capable of binding toantibody raised against a native (i.e. seen in nature) Der NP epitope.

A reagent comprising a Der NP epitope of the present invention can beused in a variety of ways in accordance with the present invention. Sucha reagent can be a desensitizing compound or a detection reagent to testfor mite allergy susceptibility or sensitivity. In one embodiment, atherapeutic composition of the present invention includes a reagentcomprising a Der NP epitope. in another embodiment, an assay kit of thepresent invention includes a reagent comprising a Der NP epitope. Oneembodiment of the present invention is a method to identify an animalsusceptible to or having an allergic response to a mite. Such a methodincludes the steps of contacting a reagent comprising a Der NP epitopewith antibodies of an animal and determining immunocomplex formationbetween the reagent and the antibodies, wherein formation of theimmunocomplex indicates that the animal is susceptible to or has saidallergic response. Another embodiment of the present invention is amethod to desensitize a host animal to an allergic response to a mite.Such a method includes the step of administering to the animal atherapeutic composition that includes a reagent comprising a Der NPepitope as a desensitizing compound.

Another embodiment of the present invention is a Der HMW-map proteinlacking Der NP epitopes. Without being bound by theory, it is believedthat such a protein would be a better desensitizing compound since sucha protein is expected to have a reduced ability to bind to IgE. Such aprotein can be produced by, for example, removing Der NP epitopes from anative Der HMW-map protein or by producing the protein recombinantly,for example in E. coli.

One embodiment of the present invention is an in vivo test that iscapable of detecting whether an animal is hypersensitive to Der HMW-mapprotein. An in vivo hypersensitivity test of the present invention isparticularly useful for identifying animals susceptible to or havingallergy to mite allergens. A suitable in vivo hypersensitivity test ofthe present invention can be, but is not limited to, a skin testcomprising administering (e.g., intradermally injecting or superficialscratching) an effective amount of a formulation containing Der HMW-mapprotein, or a mimetope thereof. Methods to conduct skin tests of thepresent invention are known to those of skill in the art and are brieflydisclosed herein.

Suitable formulations to use in an in vivo skin test include Der HMW-mapprotein, homologs of Der HMW-map protein and/or mimetopes of Der HMW-mapprotein.

It is understood by one of skill in the art that a suitable amount ofDer HMW-map protein formulation for use in a skin test of the presentinvention can vary widely depending on the allergenicity of theformulation used in the test and on the site at which the product isdelivered. Suitable amounts of Der HMW-map protein formulation for usein a skin test of the present invention include an amount capable offorming reaction, such as a detectable wheal or induration (hardness)resulting from an allergic reaction to the formulation. Preferredamounts of Der HMW-map protein for use in a skin test of the presentinvention range from about 1×10⁻⁸ micrograms (μg) to about 100 μg, morepreferably from about 1×10⁻⁷ μg to about 10 μg, and even more preferablyfrom about 1×10⁻⁶ μg to about 1 μg of Der HMW-map protein. It is to beappreciated by those of skill in the art that such amounts will varydepending upon the allergenicity of the protein being administered.

According to the present invention, Der HMW-map protein of the presentinvention can be combined with an immunopotentiator (e.g., carriers oradjuvants of the present invention as defined in detail below). A novelaspect, however, of the present invention is that Der HMW-map protein ofthe present invention can induce a hypersensitive response in theabsence of an immunopotentiator, particularly in canines.

A skin test of the present invention further comprises administering acontrol solution to an animal. A control solution can include a negativecontrol solution and/or a positive control solution. A positive controlsolution of the present invention contains an effective amount of atleast one compound known to induce a hypersensitive response whenadministered to an animal. A preferred compound for use as positivecontrol solution includes, but is not limited to, histamine. A negativecontrol solution of the present invention can comprise a solution thatis known not to induce a hypersensitive response when administered to ananimal. As such, a negative control solution can comprise a solutionhaving compounds essentially incapable of inducing a hypersensitiveresponse or simply a buffer used to prepare the formulation, such assaline. An example of a preferred negative control solution isphenolated phosphate buffered saline (available from Greer Laboratories,Inc., Lenoir, N.C.).

Hypersensitivity of an animal to one or more formulations of the presentinvention can be evaluated by measuring reactions (e.g., wheal size,induration or hardness; using techniques known to those skilled in theart) resulting from administration of one or more experimental sample(s)and control sample(s) into an animal and comparing the reactions to theexperimental sample(s) with reactions resulting from administration ofone or more control solution. Preferred devices for intradermalinjections include individual syringes. Preferred devices for scratchinginclude devices that permit the administration of a number of samples atone time. The hypersensitivity of an animal can be evaluated bydetermining if the reaction resulting from administration of aformulation of the present invention is larger than the reactionresulting from administration of a negative control, and/or bydetermining if the reaction resulting from administration of theformulation is at least about the same size as the reaction resultingfrom administration of a positive control solution. As such, if anexperimental sample produces a reaction greater than or equal to thesize of a wheal produced by administration of a positive control sampleto an animal, then that animal is hypersensitive to the experimentalsample. Conversely, if an experimental sample produces a reactionsimilar to the reaction produced by administration of a negative controlsample to an animal, then that animal is not hypersensitive to theexperimental sample.

Preferred wheal sizes for evaluation of the hypersensitivity of ananimal range from about 16 mm to about 8 mm, more preferably from about15 mm to about 9 mm, and even more preferably from about 14 mm to about10 mm in diameter.

Preferably, the ability or inability of an animal to exhibit animmediate hypersensitive response to a formulation of the presentinvention is determined by measuring wheal sizes from about 2 minutes toabout 30 minutes after administration of a sample, more preferably fromabout 10 minutes to about 25 minutes after administration of a sample,and even more preferably about 15 minutes after administration of asample.

Preferably, the ability or inability of an animal to exhibit a delayedhypersensitive response to a formulation of the present invention isdetermined by measuring induration and/or erythema from about 18 hoursto about 30 hours after administration of a sample, more preferably fromabout 20 hours to about 28 hours after administration of a sample, andeven more preferably at about 24 hours after administration of a sample.A delayed hypersensitivity response can also be measured using othertechniques such as by determining, using techniques known to those ofskill in the art, the extent of cell infiltrate at the site ofadministration during the time periods defined directly above.

In a preferred embodiment, a skin test of the present inventioncomprises intradermally injecting into an animal at a given site aneffective amount of a formulation that includes Der HMW-map protein, andintradermally injecting an effective amount of a control solution intothe same animal at a different site. It is within the scope of one ofskill in the art to use devices capable of delivering multiple samplessimultaneously at a number of sites, preferably enabling concurrentevaluation of numerous formulations. A preferred Der HMW-map protein foruse with a skin test includes full-length protein. A preferred positivecontrol sample can be a sample comprising histamine. A preferrednegative control sample can be a sample comprising diluent.

Animals suitable and preferred to test for hypersensitivity to DerHMW-map protein using a skin test of the present invention are disclosedherein. Particularly preferred animals to test with a skin test of thepresent invention include humans, canines, felines and equines, withhuman, canines and felines being even more preferred. As used herein,canine refers to any member of the dog family, including domestic dogs,wild dogs and zoo dogs. Examples of dogs include, but are not limitedto, domestic dogs, wild dogs, foxes, wolves, jackals and coyotes. Asused herein, feline refers to any member of the cat family, includingdomestic cats, wild cats and zoo cats. Examples of cats include, but arenot limited to, domestic cats, lions, tigers, leopards, panthers,cougars, bobcats, lynx, jaguars, cheetahs and servals. As used herein,equine refers to any member of the horse family, including horses,donkeys, mules and zebras.

One embodiment of the present invention is a method to detect antibodiesin vitro that bind to Der HMW-map protein (referred to herein asanti-Der HMW-map antibody) which includes the steps of; (a) contactingan isolated Der HMW-map protein with a putative anti-Der HMW-mapantibody-containing composition under conditions suitable for formationof a Der HMW-map protein:antibody complex; and (b) detecting thepresence of the antibody by detecting the Der HMW-map protein:antibodycomplex. Presence of such a Der IHMW-map protein:antibody complexindicates that the animal is producing antibody to a mite allergen.Preferred anti-Der HMW-map antibody to detect include antibodies havingan IgE or IgG isotype. Preferred anti-Der HMW-map antibody to detectinclude feline antibody, canine antibody, equine antibody and humanantibody, with feline, canine and human antibody being particularlypreferred.

As used herein, the term “contacting” refers to combining or mixing, inthis case a putative antibody-containing composition with a Der HMW-mapprotein. Formation of a complex between a Der HMW-map protein and anantibody refers to the ability of the Der HMW-map protein to selectivelybind to the antibody in order to form a stable complex that can bemeasured (i.e., detected). As used herein, the term selectively binds toan antibody refers to the ability of a Der HMW-map protein of thepresent invention to preferentially bind to an antibody, without beingable to substantially bind to other antibodies that do not specificallybind to Der HMW-map protein. Binding between a Der HMW-map protein andan antibody is effected under conditions suitable to form a complex;such conditions (e.g., appropriate concentrations, buffers,temperatures, reaction times) as well as methods to optimize suchconditions are known to those skilled in the art, and examples aredisclosed herein. Examples of complex formation conditions are alsodisclosed in, for example, in Sambrook et al., ibid.

As used herein, the term “detecting complex formation” refers todetermining if any complex is formed, i.e., assaying for the presence(i.e., existence) of a complex. If complexes are formed, the amount ofcomplexes formed can, but need not be, determined. Complex formation, orselective binding, between Der HMW-map protein and an antibody in thecomposition can be measured (i.e., detected, determined) using a varietyof methods standard in the art (see, for example, Sambrook et al.ibid.), examples of which are disclosed herein.

In one embodiment, a putative antibody-containing composition of thepresent method includes a biological sample from an animal. A suitablebiological sample includes, but is not limited to, a bodily fluidcomposition or a cellular composition. A bodily fluid refers to anyfluid that can be collected (i.e., obtained) from an animal, examples ofwhich. include, but are not limited to, blood, serum, plasma, urine,tears, aqueous humor, cerebrospinal fluid (CSF), saliva, lymph, nasalsecretions, milk and feccs. Such a composition of the present methodcan, but need not be, pretreated to remove at least some of the non-IgEor non-IgG isotypes of immunoglobulin and/or other proteins, such asalbumin, present in the fluid. Such removal can include, but is notlimited to, contacting the bodily fluid with a material, such as thelectin jacalin or an antibody that specifically binds to the constantregion of an IgA immunoglobulin (i.e., anti-IgA isotype antibody), toremove IgA antibodies and/or affinity purifying IgE or IgG antibodiesfrom other components of the body fluid by exposing the fluid to, forexample, Concanavalin A or protein G, respectively. In anotherembodiment, a composition includes collected bodily fluid that ispretreated to concentrate immunoglobulin contained in the fluid. Forexample, immunoglobulin contained in a bodily fluid can be precipitatedfrom other proteins using ammonium sulfate. A preferred composition ofthe present method is serum.

In another embodiment, an antibody-containing composition of the presentmethod includes a cell that produces IgE or IgG. Such a cell can haveIgE or IgG bound to the surface of the cell and/or can secrete IgE orIgG. An example of such a cell includes myeloma cells. IgE or IgG can bebound to the surface of a cell either directly to the membrane of thecell or bound to a molecule (e.g., an antigen) bound to the surface ofthe cell.

A complex can be detected in a variety of ways including, but notlimited to use of one or more of the following assays: an enzyme-linkedimmunoassay, a radioimmunoassay, a fluorescence immunoassay, achemiluminescent assay, a lateral flow assay, an agglutination assay, aparticulate-based assay (e.g., using particulates such as, but notlimited to, magnetic particles or plastic polymers, such as latex orpolystyrene beads), an immunoprecipitation assay, a BioCore™ assay(e.g., using colloidal gold) and an immunoblotting assay (e.g., awestern blot). Such assays are well known to those skilled in the art.Assays can be used to give qualitative or quantitative results dependingon how they are used. Some assays, such as agglutination, particulateseparation, and immunoprecipitation, can be observed visually (e.g.,either by eye or by a machines, such as a densitometer orspectrophotometer) without the need for a detectable marker.

In other assays, conjugation (i.e., attachment) of a detectable markerto the Der HMW-map protein, to antibody bound to the Der HMW-mapprotein, or to a reagent that selectively binds to the Der HMW-mapprotein or to the antibody bound to the Der HMW-map protein (describedin more detail below) aids in detecting complex formation. Examples ofdetectable markers include, but are not limited to, a radioactive label,an enzyme, a fluorescent label, a chemiluminescent label, a chromophoriclabel or a ligand. A ligand refers to a molecule that binds selectivelyto another molecule. Preferred detectable markers include, but are notlimited to, fluorescein, a radioisotope, a phosphatase (e.g., alkalinephosphatase), biotin, avidin, a peroxidase (e.g., horseradishperoxidase) and biotin-related compounds or avidin-related compounds(e.g., streptavidin or ImmunoPure® NeutrAvidin available from Pierce,Rockford, Ill.).

In one embodiment, a complex is detected by contacting a putativeantibody-containing composition with a Der HMW-map protein that isconjugated to a detectable marker. A suitable detectable marker toconjugate to a Der HMW-map protein includes, but is not limited to, aradioactive label, a fluorescent label, an enzyme label, achemiluminescent label, a chromophoric label or a ligand. A detectablemarker is conjugated to a Der HMW-map protein in such a manner as not toblock the ability of the Der HMW-map protein to bind to the antibodybeing detected.

In another embodiment, a Der HMW-map protein:antibody complex isdetected by contacting a putative antibody-containing composition with aDer HMW-map protein and then contacting the complex with an indicatormolecule. Suitable indicator molecules of the present invention includemolecules that can bind to either the Der HMW-map protein or to theantibody bound to the Der HMW-map protein. As such, an indicatormolecule can comprise, for example, an antigen and an antibody,depending upon which portion of the Der HMW-map protein;antibody complexis being detected. Preferred indicator molecules that arc antibodiesinclude, for example, anti-IgE antibodies, anti-IgG antibodies andantibodies that are known bind to Der HMW-map protein but bind to adifferent epitope on Der HMW-map protein than antibodies identified inthe putative antibody-containing composition. Preferred lectins includethose lectins that bind to high-mannose groups. An indicator moleculeitself can be attached to a detectable marker of the present invention.For example, an antibody can be conjugated to biotin, horseradishperoxidase, alkaline phosphatase or fluorescein.

In one preferred embodiment, a Der HMW-map protein:antibody complex isdetected by contacting the complex with an indicator molecule thatselectively binds to an IgE antibody (referred to herein as an anti-IgEreagent) or an IgE antibody (referred to herein as an anti-IgG reagent.Examples of such an anti-IgE or an anti-IgG antibody include, but arenot limited to, a secondary antibody that is an anti-isotype antibody(e.g., an antibody that selectively binds to the constant region of anIgE or an IgG), an antibody-binding bacterial surface protein (e.g.,Protein A or Protein G), an antibody-binding cell (e.g., a B cell, a Tcell, a natural killer cell, a polymorphonuclear leukocyte cell, amonocyte cell or a macrophage cell), an antibody-binding eukaryotic cellsurface protein (e.g., a Fc receptor), and an antibody-bindingcomplement protein. Preferred indicator molecules include, but are notlimited to, an anti-feline IgE antibody, an anti-feline IgG antibody, ananti-canine IgE antibody, an anti-canine IgG antibody, an anti-human IgEantibody, and an anti-human IgG antibody. As used herein, an anti-IgE oranti-IgG antibody includes not only a complete antibody but also anysubunit or portion thereof that is capable of selectively binding to anIgE or IgG heavy chain constant region. For example, an anti-IgE reagentor anti-IgG reagent can include an Fab fragment or a F(ab′)₂ fragment,both of which are described in detail in Janeway et al., inImmunobiology, the Immune System in Health and Disease, GarlandPublishing, Inc., NY, 1996 (which is incorporated herein by thisreference in its entirety).

In another preferred embodiment, a Der HMW-map protein:antibody complexis detected by contacting the complex with an indicator molecule thatselectively binds to Der HMW-map protein at a different epitope than theepitope at which an antibody in a putative antibody-containingcomposition binds to Der HMW-map protein.

In one embodiment a complex can be formed and detected in solution. Inanother embodiment, a complex can be formed in which one or more membersof the complex are immobilized on (e.g., coated onto) a substrate.Immobilization techniques are known to those skilled in the art.Suitable substrate materials include, but are not limited to, plastic,glass, gel, celluloid, paper, PVDF (poly-vinylidene-fluoride), nylon,nitrocellulose, and particulate materials such as latex, polystyrene,nylon, nitrocellulose, agarose and magnetic resin. Suitable shapes forsubstrate material include, but are not limited to, a well (e.g.,microtiter dish well), a plate, a dipstick, a bead, a lateral flowapparatus, a membrane, a filter, a tube, a dish, a celluloid-typematrix, a magnetic particle, and other particulates. A particularlypreferred substrate comprises an ELISA plate, a dipstick, aradioimmunoassay plate, agarose beads, plastic beads, latex beads,immunoblot membranes and immunoblot papers. In one embodiment, asubstrate, such as a particulate, can include a detectable marker.

A preferred method to detect antibody that binds to Der HMW-map proteinis an immunoabsorbent assay. An immunoabsorbent assay of the presentinvention comprises a capture molecule and an indicator molecule. Acapture molecule of the present invention binds to an IgE or an IgG insuch a manner that the IgE or IgG is immobilized to a substrate. Assuch, a capture molecule is preferably immobilized to a substrate of thepresent invention prior to exposure of the capture molecule to aputative IgE-containing composition or a putative IgG-containingcomposition. An indicator molecule of the present invention detects thepresence of an IgE or an IgG bound to a capture molecule. As such, anindicator molecule preferably is not immobilized to the same substrateas a capture molecule prior to exposure of the capture molecule to aputative IgE-containing composition or a putative IgG-containingcomposition.

A preferred immunoabsorbent assay method includes a step of either: (a)immobilizing a Der HMW-map protein on a substrate prior to contacting aDer HMW-map protein with a putative IgE-containing composition or aputative IgG-containing composition to form a Der HMW-map protein-immobilized substrate; and (b) binding a putative IgE-containingcomposition or a putative IgG-containing composition on a substrateprior to contacting Der HMW-map protein with a putative IgE-containingcomposition or a putative IgG-containing composition, to form a putativeIgE-containing composition-bound substrate or a putative IgG-containingcomposition-bound substrate, respectively. Preferably, the substrateincludes a non-coated substrate, a Der HMW-map protein-immobilizedsubstrate, an anti-IgE antibody-immobilized substrate or anti-IgGantibody-immobilized substrate.

Both a capture molecule and an indicator molecule of the presentinvention are capable of binding to an IgE, an IgG or Der HMW-mapprotein. Preferably, a capture molecule binds to a different region ofan IgE, an IgG or Der HMW-map protein than an indicator molecule,thereby allowing a capture molecule to be bound to an IgE, an IgG or DerHMW-map protein at the same time as an indicator molecule. The use of areagent as a capture molecule or an indicator molecule depends uponwhether the molecule is immobilized to a substrate when the molecule isexposed to an IgE, an IgG or Der HMW-map protein. For example, a DerHMW-map protein of the present invention is used as a capture moleculewhen the Der HMW-map protein is bound on a substrate. Alternatively, aDer HMW-map protein is used as an indicator molecule when the DerHMW-map protein is not bound on a substrate. Suitable molecules for useas capture molecules or indicator molecules include, but are not limitedto, a Der HMW-map protein of the present invention, an anti-IgE antibodyreagent or an anti-IgG antibody reagent of the present invention.

An immunoabsorbent assay of the present invention can further compriseone or more layers and/or types of secondary molecules or other bindingmolecules capable of detecting the presence of an indicator molecule.For example, an untagged (i.e., not conjugated to a detectable marker)secondary antibody that selectively binds to an indicator molecule canbe bound to a tagged (i.e., conjugated to a detectable marker) tertiaryantibody that selectively binds to the secondary antibody. Suitablesecondary antibodies, tertiary antibodies and other secondary ortertiary molecules can be selected by those of skill in the art.Preferred secondary molecules of the present invention include anantigen, an anti-IgE idiotypic antibody (i.e., an antibody that binds toan epitope unique to the anti-IgE antibody), an anti-IgE isotypicantibody, an anti-IgG idiotypic antibody (i.e., an antibody that bindsto an epitope unique to the anti-IgG antibody), and an anti-IgG isotypicantibody. Preferred tertiary molecules can be selected by a skilledartisan based upon the characteristics of the secondary molecule. Thesame strategy is applied for subsequent layers.

In one embodiment, Der HMW-map protein is used as a capture molecule bybeing immobilized on a substrate, such as a microtiter dish well or adipstick. A biological sample collected from an animal is applied to thesubstrate and incubated under conditions suitable (i.e., sufficient) toallow for Der HMW-map protein:antibody complex formation bound to thesubstrate (i.e., IgE or IgG in a sample binds to Der HMW-map proteinimmumobilized on a substrate). Excess non-bound material (i.e., materialfrom the biological sample that has not bound to the Der HMW-mapprotein), if any, is removed from the substrate under conditions thatretain antigen:antibody complex binding to the substrate. Preferredconditions are generally disclosed in Sambrook et al., ibid. Anindicator molecule that can selectively bind to an IgE or an IgG boundto the antigen is added to the substrate and incubated to allowformation of a complex between the indicator molecule and the DerHMW-map protein:antibody complex. Excess indicator molecule is removed,a developing agent is added if required, and the substrate is submittedto a detection device for analysis. A preferred indicator molecule forthis embodiment is an anti-IgG antibody to detect IgG antibody bound toDer HMW-map protein or an anti-IgE antibody to detect IgE antibody boundto Der HMW-map protein. Preferably the anti-IgG or anti-IgE antibody areconjugated to biotin, to a fluorescent label or to an enzyme label.

In one embodiment, an anti-IgE or anti-IgG antibody (e.g., isotype oridiotype specific antibody) is used as a capture molecule by beingimmobilized on a substrate, such as a microtiter dish well or adipstick: A biological sample collected from an animal is applied to thesubstrate and incubated under conditions suitable to allow for anti-IgEantibody:IgE complex or anti-IgG antibody:IgG complex formation,respectively, bound to the substrate. Excess non-bound material, if any,is removed from the substrate under conditions that retain anti-IgEantibody:IgE complex or anti-IgG antibody:IgG complex binding to thesubstrate. Der HMW-map protein is added to the substrate and incubatedto allow formation of a complex between the Der HMW-map protein and theanti-IgE antibody:IgE complex or anti-IgG antibody:IgG complex.Preferably, the Der HMW-map protein is conjugated to a detectable marker(preferably to biotin, an enzyme label or a fluorescent label). ExcessDer HMW-map protein is removed, a developing agent is added if required,and the substrate is submitted to a detection device for analysis.

In one embodiment, an immunoabsorbent assay of the present inventiondoes not utilize a capture molecule. In this embodiment, a biologicalsample collected from an animal is applied to a substrate, such as amicrotiter dish well or a dipstick, and incubated under conditionssuitable to allow for IgE or IgG binding to the substrate. Any IgE orIgG present in the bodily fluid is immobilized on the substrate. Excessnon-bound material, if any, is removed from the substrate underconditions that retain IgE or IgG binding to the substrate. Der HMW-mapprotein is added to the substrate and incubated to allow formation of acomplex between the Der HMW-map protein and the IgE or IgG. Preferably,the Der HMW-map protein is conjugated to a detectable marker (preferablyto biotin, an enzyme label or a fluorescent label). Excess Der HMW-mapprotein is removed, a developing agent is added if required, and thesubstrate is submitted to a detection device for analysis.

Another preferred method to detect IgE or IgG is a lateral flow assay,examples of which are disclosed in U.S. Pat. No. 5,424,193, issued Jun.13, 1995, by Pronovost et al.; U.S. Pat. No. 5,415,994, issued May 16,1995, by Imrich et al; WO 94/29696, published Dec. 22, 1994, by Milleret al.; and WO 94/01775, published Jan. 20, 1994, by Pawlak et al.; eachof these patent publications is incorporated by reference herein in itsentirety. In one embodiment, a biological sample is placed in a lateralflow apparatus that includes the following components: (a) a supportstructure defining a flow path; (b) a labeling reagent comprising a beadconjugated to Der HMW-map protein, the labeling reagent beingimpregnated within the support structure in a labeling zone; and (c) acapture reagent comprising an IgE-binding or an IgG-binding composition.The capture reagent is located downstream of the labeling reagent withina capture zone fluidly connected to the labeling zone in such a mannerthat the labeling reagent can flow from the labeling zone into thecapture zone. The support structure comprises a material that does notimpede the flow of the beads from the labeling zone to the capture zone.Suitable materials for use as a support structure include ionic (i.e.,anionic or cationic) material. Examples of such a material include, butare not limited to, nitrocellulose (NC), PVDF, carboxymethylcellulose(CM). The support structure defines a flow path that is lateral and isdivided into zones, namely a labeling zone and a capture zone. Theapparatus can further comprise a sample receiving zone located along theflow path, more preferably upstream of the labeling reagent. The flowpath in the support structure is created by contacting a portion of thesupport structure downstream of the capture zone, preferably at the endof the flow path, to an absorbent capable of absorbing excess liquidfrom the labeling and capture zones.

In this embodiment, the biological sample is applied to the samplereceiving zone which includes a portion of the support structure. Thelabeling zone receives the sample from the sample receiving zone whichis directed downstream by the flow path. The labeling zone comprises thelabeling reagent that binds to IgE or IgG, or both. A preferred labelingreagent is Der HMW-map protein conjugated, either directly or through alinker, to a plastic bead substrate, such as to a latex bead. Thesubstrate also includes a detectable marker, preferably a colorimetricmarker. Typically, the labeling reagent is impregnated to the supportstructure by drying or lyophilization. The sample structure alsocomprises a capture zone downstream of the labeling zone. The capturezone receives labeling reagent from the labeling zone which is directeddownstream by the flow path. The capture zone contains the capturereagent, in this case an anti-IgE or anti-IgG antibody, or both, asdisclosed above, that immobilizes the IgE and/or IgG complexed to theDer HMW-map protein in the capture zone. The capture reagent ispreferably fixed to the support structure by drying or lyophilizing. Thelabeling reagent accumulates in the capture zone and the accumulation isassessed visually or by an optical detection device.

In another embodiment, a lateral flow apparatus used to detect IgE orIgG includes: (a) a support structure defining a flow path; (b) alabeling reagent comprising an anti-IgE or an anti-IgG antibody, orboth, as described above, the labeling reagent impregnated within thesupport structure in a labeling zone; and (c) a capture reagentcomprising Der HMW-map protein, the capture reagent being locateddownstream of the labeling reagent within a capture zone fluidlyconnected to the labeling zone in such a manner that the labelingreagent can flow from the labeling zone into the capture zone. Theapparatus preferably also includes a sample receiving zone located alongthe flow path, preferably upstream of the labeling reagent. Theapparatus preferably also includes an absorbent located at the end ofthe flow path.

An animal hypersensitive to Der HMW-map protein is identified bycomparing the level of immunocomplex formation using samples of bodyfluid with the level of immunocomplex formation using control samples.An immunocomplex refers to a complex comprising an antibody and DerHMW-map protein (i.e., Der HMW-map protein:antibody complex). As such,immunocomplexes form using positive control samples and do not formusing negative control samples. As such, if a body fluid sample resultsin immunocomplex formation greater than or equal to immunocomplexformation using a positive control sample, then the animal from whichthe fluid was taken is hypersensitive to the Der HMW-map protein boundto the substrate. Conversely, if a body fluid sample results inimmunocomplex formation similar to immunocomplex formation using anegative control sample, then the animal from which the fluid was takenis not hypersensitive to the Der HMW-map protein bound to the substrate.

It is within the scope of the present invention that two or moredifferent skin tests and/or in vitro tests can be used in combinationfor diagnostic purposes. For example, the immediate hypersensitivity ofan animal to Der HMW-map protein can be tested using an in vitroimmunoabsorbent test capable of detecting IgE antibodies specific forDer HMW-map protein in the animal's bodily fluid. While most animalsthat display delayed hypersensitivity to Der HMW-map protein alsodisplay immediate hypersensitivity to the allergen, a small number ofanimals that display delayed hypersensitivity to an allergen do notdisplay immediate hypersensitivity to the allergen. In such cases,following negative results from the IgE-specific in vitro test, thedelayed hypersensitivity of the animal to Der HMW-map protein can betested using an skin test of the present invention.

The present invention also includes kits to detect antibodies that bindspecifically to Der HMW-map protein based on each of the discloseddetection methods. One embodiment is a kit to detect Der HMW-mapprotein-specific antibodies comprising Der HMW-map protein and a meansfor detecting an IgE and/or an IgG. Suitable means of detection includecompounds disclosed herein that bind to either the Der HMW-map proteinor to an IgE and/or an IgG. A preferred kit of the present inventionfurther comprises a detection means including an antibody capable ofselectively binding to an IgE or IgG disclosed herein and/or a compoundcapable of binding to a detectable marker conjugated to a Der HMW-mapprotein (e.g., avidin, streptavidin and ImmunoPure® NeutrAvidin when thedetectable marker is biotin).

Another preferred kit of the present invention is an allergen kitcomprising Der HMW-map protein and an allergen commonly detected in thesame environment as mite allergen. Suitable and preferred mite-relatedallergens for use with the present kit include those mite-relatedallergens disclosed herein.

A preferred kit of the present invention includes those in which DerHMW-map protein is immobilized on a substrate. If a kit comprises DerHMW-map protein and another allergen, the kit can comprise one or morecompositions, each composition comprising one allergen. As such, eachallergen can be tested separately. A kit can also contain two or morediagnostic reagents for IgE or IgG, or other compounds as disclosedherein. Particularly preferred are kits used in a lateral flow assayformat. It is within the scope of the present invention that a lateralflow assay kit can include one or more lateral flow assay apparatuses.Multiple lateral flow apparatuses can be attached to each other at oneend of each apparatus, thereby creating a fan-like structure.

Another aspect of the present invention includes treating animalssusceptible to or having mite allergy, with a Der HMW-map proteinformulation of the present invention. According to the presentinvention, the term treatment can refer to the regulation of ahypersensitive response by an animal to mite allergens. Regulation caninclude, for example, immunomodulation of cells involved in the animal'shypersensitive response. Immunomodulation can include modulating theactivity of molecules typically involved in an immune response (e.g.,antibodies, antigens, major histocompatibility molecules (MHC) andmolecules co-reactive with MHC molecules). In particular,immunomodulation refers to modulation of antigen:antibody interactionsresulting in inflammatory responses, immunosuppression, andimmunotolerization of cells involved in a hypersensitive response.Immunosuppression refers to inhibiting an immune response by, forexample, killing particular cells involved in the immune response.Immunotolerization refers to inhibiting an immune response by anergizing(i.e., diminishing reactivity of a T cell to an antigen) particularcells involved in the immune response.

One embodiment of the present invention is a therapeutic compositionthat includes desensitizing compounds capable of inhibiting an immuneresponse to Der HMW-map protein of the present invention. Suchdesensitizing compounds include blocking compounds, toleragens and/orsuppressor compounds. Blocking compounds comprise compounds capable ofmodulating antigen:antibody interactions that can result in inflammatoryresponses, toleragens are compounds capable of immunotolerizing ananimal, and suppressor compounds are capable of immunosuppressing ananimal. A desensitizing compound of the present invention can be solubleor membrane-bound. Membrane-bound desensitizing compounds can beassociated with biomembranes, including cells, liposomes, planarmembranes or micelles. A soluble desensitizing compound of the presentinvention is useful for: (1) inhibiting a Type I hypersensitivityreaction by blocking IgE:antigen mediated de-granulation of mast cells;(2) inhibiting a Type III hypersensitivity reaction by blockingIgG:antigen complex formation leading to complement destruction ofcells; and (3) inhibiting a Type IV hypersensitivity reaction byblocking T helper cell stimulation of cytokine secretion by macrophages.A membrane-bound desensitizing compound of the present invention isuseful for: (1) inhibiting a Type II hypersensitivity reaction byblocking IgG:antigen complex formation on the surface of cells leadingto complement destruction of cells; (2) inhibiting a Type IIhypersensitivity reaction by blocking IgG regulated signal transductionin immune cells; and (3) inhibiting a Type IV hypersensitivity reactionby blocking T cytotoxic cell killing of antigen-bearing cells. Examplesof desensitizing compounds include, but are not limited to, muteins,mimetopes and antibodies of the present invention, as well as otherinhibitors of the present invention that inhibit binding between aprotein of the present invention and IgE.

A desensitizing compound of the present invention can also be covalentlylinked to a ligand molecule capable of targeting the desensitizingcompound to a specific cell involved in a hypersensitive response to DerHMW-map protein. Appropriate ligands with which to link a desensitizingcompound include, for example, at least a portion of an immunoglobulinmolecule, cytokines, lectins, heterologous allergens, CD8 molecules ormajor histocompatibility molecules (e.g., MHC class I or MHC class IImolecules). Preferred portions of immunoglobulin molecules to link to adesensitizing compound include variable regions capable of binding toimmune cell specific surface molecules and constant regions capable ofbinding to Fc receptors on immune cells, in particular IgE constantregions. Preferred CD8 molecules include at least the extracellularfunctional domains of the a chain of CD8. An immune cell refers to acell involved in an immune response, in particular, cells having MHCclass I or MHC class II molecules. Preferred immune cells includeantigen presenting cells, T cells and B cells.

In one embodiment, a therapeutic composition of the present inventionincludes Der HMW-map protein of the present invention, a mimetope ormutein thereof, or a Der HMW-map nucleic acid molecule of the presentinvention. Suitable therapeutic compositions of the present inventionfor treating mite allergy include Der HMW-map protein, a mimetope ormutein thereof, or a Der HMW-map nucleic acid molecule of the presentinvention. Preferred therapeutic compositions include: an isolated miteallergenic protein encoded a nucleic acid molecule that hybridizes understringent hybridization conditions with the complement of a nucleic acidmolecule that encodes an amino acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:18,SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:38,SEQ ID NO:41, and SEQ ID NO:44; a mimetope of the mite allergenicprotein; a mutein of the mite allergenic protein; and an isolatednucleic acid molecule selected from the group consisting of: a nucleicacid molecule comprising at least about 150 nucleotides, wherein saidnucleic acid molecule comprising at least about 150 nucleotideshybridizes, in a solution comprising 1×SSC and 0% formamide, at atemperature of about 50° C., to a nucleic acid sequence selected fromthe group consisting of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:34, SEQ ID NO:36, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ IDNO:45, and a nucleic acid sequence encoding a protein comprising theamino acid sequence SEQ ID NO:33 and a complement thereof, and a nucleicacid molecule comprising a fragment of any of said nucleic acidmolecules comprising at least about 150 nucleotides. A preferred DerHMW-map mutein comprises at least a portion of Der HMW-map protein, inwhich a suitable number of cysteine residues have been removed orreplaced with a non-cysteine residue such that the altered Der HMW-mapprotein is not toxic to an animal (e.g., does not cause anaphylaxis).

In another embodiment, a therapeutic composition of the presentinvention includes a nucleic acid molecule encoding a Der HMW-mapprotein that can be administered to an animal in a fashion to enableexpression of that nucleic acid molecule into a Der HMW-map protein inthe animal. Nucleic acid molecules can be delivered to an animal in avariety of methods including, but not limited to, (a) administering anaked (i.e., not packaged in a viral coat or cellular membrane) nucleicacid molecule (e.g., as naked DNA or RNA molecules, such as is taught,for example in Wolff et al., 1990, Science 247, 1465-1468) or (b)administering a nucleic acid molecule packaged as a recombinant virus oras a recombinant cell (i.e., the nucleic acid molecule is delivered by aviral or cellular vehicle).

A naked nucleic acid molecule of the present invention includes anucleic acid molecule of the present invention and preferably includes arecombinant molecule of the present invention that preferably isreplication, or otherwise amplification, competent. A naked nucleic acidof the present invention can comprise one or more nucleic acid moleculesof the present invention in the form of, for example, a bicistronicrecombinant molecule having, for example one or more internal ribosomeentry sites. Preferred naked nucleic acid molecules include at least aportion of a viral genome (i.e., a viral vector). Preferred viralvectors include those based on alphaviruses, poxviruses, adenoviruses,herpesviruses, picomaviruses, and retroviruses, with those based onalphaviruses (such as Sindbis or Semliki virus), species-specificherpesviruses and species-specific poxviruses being particularlypreferred. Any suitable transcription control sequence can be used,including those disclosed as suitable for protein production.Particularly preferred transcription control sequence includecytomegalovirus intermediate early (preferably in conjunction withIntron-A), Rous Sarcoma Virus long terminal repeat, and tissue-specifictranscription control sequences, as well as transcription controlsequences endogenous to viral vectors if viral vectors are used. Theincorporation of “strong” poly(A) sequences are also preferred.

Naked nucleic acid molecules of the present invention can beadministered by a variety of methods. Suitable delivery methods include,for example, intramuscular injection, subcutaneous injection,intradermal injection, intradermal scarification, particle bombardment,oral application, and nasal application, with intramuscular injection,intradermal injection, intraderrnal scarification and particlebombardment being preferred, and intramuscular injection being even morepreferred. A preferred single dose of a naked DNA molecule ranges fromabout 1 nanogram (ng) to about 1 milligram (mg), depending on the routeof administration and/or method of delivery, as can be determined bythose skilled in the art. Examples of administration methods aredisclosed, for example, in U.S. Pat. No. 5,204,253, by Bruner, et al.,issued Apr. 20, 1993, PCT Publication No. WO 95/19799, published Jul.27, 1995, by McCabe, and PCT Publication No. WO 95/05853, published Mar.2, 1995, by Carson, et al. Naked DNA molecules of the present inventioncan be contained in an aqueous excipient (e.g., phosphate bufferedsaline) and/or with a carrier (e.g., lipid-based vehicles), or it can bebound to microparticles (e.g., gold particles).

A recombinant virus of the present invention includes a recombinantmolecule of the present invention that is packaged in a viral coat andthat can be expressed in an animal after administration. Preferably, therecombinant molecule is packaging-deficient and/or encodes an attenuatedvirus. A number of recombinant viruses can be used, including, but notlimited to, those based on alphaviruses, poxviruses, adenoviruses,herpesviruses, picornaviruses and retroviruses. Preferred recombinantviruses are those based on alphaviruses (such as Sindbis virus), raccoonpoxviruses, species-specific herpesviruses and species-specificpoxviruses. An example of methods to produce and use alphavirusrecombinant virus is disclosed in PCT Publication No. WO 94/17813, byXiong et al., published Aug. 18, 1994, which is incorporated byreference herein in its entirety.

When administered to an animal, a recombinant virus of the presentinvention infects cells within the recipient animal and directs theproduction of a protein or RNA nucleic acid molecule that is capable ofreducing Der HMW-map protein-mediated biological responses in theanimal. For example, a recombinant virus comprising a Der HMW-mapnucleic acid molecule of the present invention is administered accordingto a protocol that results in the animal producing an amount of proteinor RNA sufficient to reduce Der HMW-map protein-mediated biologicalresponses. A preferred single dose of a recombinant virus of the presentinvention is from about 1×10⁴ to about 1×10⁷ virus plaque forming units(pfu) per kilogram body weight of the animal. Administration protocolsare similar to those described herein for protein-based compositions,with subcutaneous, intramuscular, intranasal and oral administrationroutes being preferred.

A recombinant cell vaccine of the present invention includes recombinantcells of the present invention that express at least one protein of thepresent invention. Preferred recombinant cells for this embodimentinclude Salmonella, E. coli, Listeria, Mycobacterium, S. frugiperda,yeast, (including Saccharomyces cerevisiae and Pichia pastoris), BHK,CV-1, myoblast G8, COS (e.g., COS-7), Vero, MDCK and CRFK recombinantcells. Recombinant cell vaccines of the present invention can beadministered in a variety of ways but have the advantage that they canbe administered orally, preferably at doses ranging from about 10⁸ toabout 10¹² cells per kilogram body weight. Administration protocols aresimilar to those described herein for protein-based vaccines.Recombinant cell vaccines can comprise whole cells, cells stripped ofcell walls or cell lysates.

The efficacy of a therapeutic composition of the present invention todesensitize an animal against mite allergy can be tested in a variety ofways including, but not limited to, using in vivo skin test methodsdisclosed herein, detection of cellular immunity activity in the treatedanimal, or determine levels of IgE that bind specifically to a DerHMW-map protein of the present invention. Methods to determine cellularimmunity activity and IgE levels in an animal are known to those ofskill in the art. In one embodiment, therapeutic compositions can betested in animal models such as dogs, cats, rabbits and mice, and canalso be tested in humans. Such techniques are known to those skilled inthe art.

Preferred nucleic acid molecules to use with a therapeutic compositionof the present invention include any Der HMW-map nucleic acid moleculedisclosed herein, in particular SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:43, SEQ ID NO:45 and/or a nucleic acid sequence encoding a proteincomprising the amino acid sequence SEQ ID NO:33 and a complementthereof.

A recombinant cell useful in a therapeutic composition of the presentinvention includes recombinant cells of the present invention thatcomprises Der HMW-map protein of the present invention. Preferredrecombinant cells for this embodiment include Salmonella, E. coli,Listeria, Mycobacterium, S. frugiperda, yeast, (including Saccharomycescerevisiae), BHK, CV-1, myoblast G8, COS (e.g., COS-7), Vero, MDCK andCRFK recombinant cells. A recombinant cell of the present invention canbe administered in a variety of ways but have the advantage that theycan be administered orally, preferably at doses ranging from about 10⁸to about 10¹² cells per kilogram body weight. Administration protocolsare similar to those described herein for protein compositions.Recombinant cells can comprise whole cells, cells stripped of cell wallsor cell lysates.

One embodiment of the present invention is a method of immunotherapycomprising administering to an animal an effective amount of atherapeutic composition comprising a Der HMW-map protein of the presentinvention. Suitable therapeutic compositions and methods ofadministration are disclosed herein. According to the present invention,a therapeutic composition and method of the present invention can beused to prevent or alleviate symptoms associated with mite allergenpathogenesis.

The efficacy of a therapeutic composition of the present invention toeffect an allergic response to Der HMW-map protein can be tested usingstandard methods for detecting Der HMW-map protein-mediated immunityincluding, but not limited to, immediate hypersensitivity, delayedhypersensitivity, antibody-dependent cellular cytotoxicity (ADCC),immune complex activity, mitogenic activity, histamine release assaysand other methods such as those described in Janeway et al., ibid.

The present invention also includes a therapeutic composition comprisingone or more therapeutic compounds of the present invention. Examples ofsuch therapeutic compounds include, for example, other allergensdisclosed herein.

Therapeutic compositions of the present invention can be formulated inan excipient that the animal to be treated can tolerate. Examples ofsuch excipients include water, saline, Ringer's solution, dextrosesolution, Hank's solution, and other aqueous physiologically balancedsalt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil,ethyl oleate, or triglycerides may also be used. Other usefulformulations include suspensions containing viscosity enhancing agents,such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipientscan also contain minor amounts of additives, such as substances thatenhance isotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer and Tris buffer, while examples ofpreservatives include thimerosal, o-cresol, formalin and benzyl alcohol.Standard formulations can either be liquid injectables or solids whichcan be taken up in a suitable liquid as a suspension or solution forinjection. Thus, in a non-liquid formulation, the excipient can comprisedextrose, human serum albumin, preservatives, etc., to which sterilewater or saline can be added prior to administration.

In one embodiment of the present invention, a therapeutic compositioncan include an adjuvant. Adjuvants are agents that are capable ofenhancing the immune response of an animal to a specific antigen.Suitable adjuvants include, but are not limited to, cytokines,chemokines, and compounds that induce the production of cytokines andchemokines (e.g., granulocyte macrophage colony stimulating factor(GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophagecolony stimulating factor (M-CSF), colony stimulating factor (CSF),Flt-3 ligand, erythropoietin (EPO), interleukin 2 (IL-2), interleukin-3(IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6(IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 10(IL-10), interleukin 12 (IL- 12), interferon gamma, interferon gammainducing factor I (IGIF), transforming growth factor beta, RANTES(regulated upon activation, normal T cell expressed and presumablysecreted), macrophage inflammatory proteins (e.g., MIP-1 alpha and MIP-1beta), and Leishmania elongation initiating factor (LEIF); bacterialcomponents (e.g., endotoxins, in particular superantigens, exotoxins andcell wall components); aluminum-based salts; calcium-based salts;silica; polynucleotides; toxoids; serum proteins, viral coat proteins;block copolymer adjuvants (e.g., Hunter's Titermax™ adjuvant (Vaxcel™,Inc. Norcross, Ga.), Ribi adjuvants (Ribi ImmunoChem Research, Inc.,Hamilton, Mont.);. and saponins and their derivatives (e.g., Quil A(Superfos Biosector A/S, Denmark). Protein adjuvants of the presentinvention can be delivered in the form of the protein themselves or ofnucleic acid molecules encoding such proteins using the methodsdescribed herein.

In one embodiment of the present invention, a therapeutic compositioncan include a carrier. Carriers include compounds that increase thehalf-life of a therapeutic composition in the treated animal. Suitablecarriers include, but are not limited to, polymeric controlled releasevehicles, biodegradable implants, liposomes, bacteria, viruses, othercells, oils, esters, and glycols.

One embodiment of the present invention is a controlled releaseformulation that is capable of slowly releasing a composition of thepresent invention into an animal. As used herein, a controlled releaseformulation comprises a composition of the present invention in acontrolled release vehicle. Suitable controlled release vehiclesinclude,, but are not limited to, biocompatible polymers, otherpolymeric matrices, capsules, microcapsules, microparticles, boluspreparations, osmotic pumps, diffusion devices, liposomes, lipospheres,and transdermal delivery systems. Other controlled release formulationsof the present invention include liquids that, upon administration to ananimal, form a solid or a gel in situ. Preferred controlled releaseformulations are biodegradable (i.e., bioerodible).

A preferred controlled release formulation of the present invention iscapable of releasing a therapeutic composition of the present inventioninto the blood of an animal at a constant rate sufficient to attaintherapeutic dose levels of the composition to reduce mite allergy in theanimal. As used herein, mite allergy refers to cellular responses thatoccur when mite allergens contact an animal. For example, IgE thatspecifically binds to mite allergen becomes coupled with Fc epsilonreceptor, resulting in Fc epsilon receptor-mediated biological responseincluding release of biological mediators, such as histamine,prostaglandins and/or proteases, that can trigger clinical symptoms ofallergy. The therapeutic composition is preferably released over aperiod of time ranging from about 1 to about 12 months. A preferredcontrolled release formulation of the present invention is capable ofeffecting a treatment preferably for at least about 1 month, morepreferably for at least about 3 months, even more preferably for atleast about 6 months, even more preferably for at least about 9 months,and even more preferably for at least about 12 months.

Therapeutic compositions of the present invention can be sterilized byconventional methods which do not result in protein degradation (e.g.,filtration) and/or lyophilized.

A therapeutic composition of the present invention can be administeredto any animal susceptible to mite allergy as herein described.Acceptable protocols by which to administer therapeutic compositions ofthe present invention in an effective manner can vary according toindividual dose size, number of doses, frequency of dose administration,and mode of administration. Determination of such protocols can beaccomplished by those skilled in the art. An effective dose refers to adose capable of treating an animal against hypersensitivity to miteallergens. Effective doses can vary depending upon, for example, thetherapeutic composition used and the size and type of the recipientanimal. Effective doses to immunomodulate an animal against miteallergens include doses administered over time that are capable ofalleviating a hypersensitive response by an animal to mite allergens.For example, a first tolerizing dose can comprise an amount of atherapeutic composition of the present invention that causes a minimalhypersensitive response when administered to a hypersensitive animal. Asecond tolerizing dose can comprise a greater amount of the sametherapeutic composition than the first dose. Effective tolerizing dosescan comprise increasing concentrations of the therapeutic compositionnecessary to tolerize an animal such that the animal does not have ahypersensitive response to exposure to mite allergens. An effective doseto desensitize an animal can comprise a concentration of a therapeuticcomposition of the present invention sufficient to block an animal fromhaving a hypersensitive response to exposure to a mite allergen presentin the environment of the animal. Effective desensitizing doses caninclude repeated doses having concentrations of a therapeuticcomposition that cause a minimal hypersensitive response whenadministered to a hypersensitive animal.

A suitable single dose is a dose that is capable of treating an animalagainst hypersensitivity to mite allergens when administered one or moretimes over a suitable time period. For example, a preferred single doseof a mite allergen, or mimetope therapeutic composition is from about0.5 ng to about 1 g of the therapeutic composition per kilogram bodyweight of the animal. Further treatments with the therapeuticcomposition can be administered from about 1 day to 1 year after theoriginal administration. Further treatments with the therapeuticcomposition preferably are administered when the animal is no longerprotected from hypersensitive responses to mite allergens. Particularadministration doses and schedules can be developed by one of skill inthe art based upon the parameters discussed above. Modes ofadministration can include, but are not limited to, subcutaneous,intradermal, intravenous, nasal, oral, transdermal and intramuscularroutes.

A therapeutic composition of the present invention can be used inconjunction with other compounds capable of modifying an animal'shypersensitivity to mite allergens. For example, an animal can betreated with compounds capable of modifying the function of a cellinvolved in a hypersensitive response, compounds that reduce allergicreactions, such as by systemic agents or anti-inflammatory agents (e.g.,anti-histamines, anti-steroid reagents, anti-inflammatory reagents andreagents that drive immunoglobulin heavy chain class switching from IgEto IgG). Suitable compounds useful for modifying the function of a cellinvolved in a hypersensitive response include, but are not limited to,antihistamines, cromolyn sodium, theophylline, cyclosporin A, adrenalin,cortisone, compounds capable of regulating cellular signal transduction,compounds capable of regulating adenosine 3′,5′-cyclic phosphate (cAMP)activity, and compounds that block IgE activity, such as peptides fromIgE or IgE specific Fc receptors, antibodies specific for peptides fromIgE or IgE-specific Fc receptors, or antibodies capable of blockingbinding of IgE to Fc receptors.

Compositions of the present invention can be administered to any animalhaving or susceptible to mite allergen hypersensitivity. Preferredanimals to treat include mammals and birds, with felines, canines,equines, humans and other pets, work and/or economic food animals.Particularly preferred animals to protect are felines and canines.

Another aspect of the present invention includes a method forprescribing treatment for animals susceptible to or havinghypersensitivity to mite allergens, using a formulation of the presentinvention. A preferred method for prescribing treatment for miteallergen hypersensitivity, for example, comprises: (1) intradermallyinjecting into an animal at one site an effective amount of aformulation containing a mite allergen of the present invention, or amimetope thereof (suitable and preferred formulations are disclosedherein); (2) intradermally injecting into the animal at a second site aneffective amount of a control solution; (3) evaluating if the animal hasmite allergen hypersensitivity by measuring and comparing the wheal sizeresulting from injection of the formulation with the wheal sizeresulting from injection of the control solution; and (4) prescribing atreatment for the mite allergen hypersensitivity.

An alternative preferred method for prescribing treatment for miteallergen hypersensitivity comprises: (1) contacting a first portion of asample of bodily fluid obtained from an animal to be tested with aneffective amount of a formulation containing mite allergen, or amimetope thereof (suitable and preferred formulations are disclosedherein) to form a first immunocomplex solution; (2) contacting apositive control antibody to form a second immunocomplex solution; (3)evaluating if the animal has mite allergen hypersensitivity by measuringand comparing the amount of immunocomplex formation in the first andsecond immunocomplex solutions; and (4) prescribing a treatment for themite allergen hypersensitivity. It is to be noted that similar methodscan be used to prescribe treatment for allergies using mite allergenformulations as disclosed herein.

Another aspect of the present invention includes a method for monitoringanimals susceptible to or having mite allergen hypersensitivity, using aformulation of the present invention. In vivo and in vitro tests of thepresent invention can be used to test animals for mite allergenhypersensitivity prior to and following any treatment for mite allergenhypersensitivity. A preferred method to monitor treatment of miteallergen hypersensitivity (which can also be adapted to monitortreatment of other allergies) comprises: (1) intradermally injecting ananimal at one site with an effective amount of a formulation containingmite allergen, or a mimetope thereof (suitable and preferredformulations are disclosed herein); (2) intradermally injecting aneffective amount of a control solution into the animal at a second site;and (3) determining if the animal is desensitized to mite allergens bymeasuring and comparing the wheal size resulting from injection of theformulation with the wheal size resulting from injection of the controlsolution.

An alternative preferred method to monitor treatment of mite allergenhypersensitivity (which can be adapted to monitor treatments of otherallergies) comprises: (1) contacting a first portion of a sample ofbodily fluid obtained from an animal to be tested with an effectiveamount of a formulation containing a mite allergen or mimetope thereof(suitable and preferred formulations are disclosed herein) to form afirst immunocomplex solution; (2) contacting a positive control antibodyto form a second immunocomplex solution; and (3) determining if theanimal is desensitized to mite allergens by measuring and comparing theamount of immunocomplex formation in the first and second immunocomplexsolutions.

The present invention also includes antibodies capable of selectivelybinding to mite allergen, or mimetope thereof. Such an antibody isherein referred to as an anti-mite allergen antibody. As used herein,the term “selectively binds to” refers to the ability of such anantibody to preferentially bind to mite allergens and mimetopes thereof.In particular, the present invention includes antibodies capable ofselectively binding to Der HMW-map protein. Binding can be measuredusing a variety of methods known to those skilled in the art includingimmunoblot assays, immunoprecipitation assays, enzyme immunoassays(e.g., ELISA), radioimmunoassays, immunofluorescent antibody assays andimmunoelectron microscopy; see, for example, Sambrook et al., ibid.

Antibodies of the present invention can be either polyclonal ormonoclonal antibodies. Antibodies of the present invention includefunctional equivalents such as antibody fragments andgenetically-engineered antibodies, including single chain antibodies,that are capable of selectively binding to at least one of the epitopesof the protein or mimetope used to obtain the antibodies. Preferredantibodies are raised in response to Der HMW-map proteins, or mimetopesthereof. More preferred Der HMW-map protein against which to raise anantibody includes at least a portion of a protein having the amino acidsequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:g, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:18,SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:38,SEQ ID NO:41, and/or SEQ ID NO:44, or homologs thereof. Preferably, anantibody of the present invention has a single site binding affinity offrom about 10³M⁻¹ to about 10¹²M⁻¹ for a Der HMW-map protein of thepresent invention.

A preferred method to produce antibodies of the present inventionincludes administering to an animal an effective amount of a Der HMW-mapprotein or mimetope thereof to produce the antibody and recovering theantibodies. Antibodies raised against defined products or mimetopes canbe advantageous because such antibodies are not substantiallycontaminated with antibodies against other substances that mightotherwise cause interference in a diagnostic assay or side effects ifused in a therapeutic composition.

Antibodies of the present invention have a variety of potential usesthat are within the scope of the present invention. For example, suchantibodies can be used (a) as vaccines to passively immunize an animalin order to protect the animal from mite allergen hypersensitivity, (b)as positive controls in test kits, and/or (c) as tools to recoverdesired mite allergens from a mixture of proteins and othercontaminants.

The following examples are provided for the purposes of illustration andare not intended to limit the scope of the present invention.

EXAMPLES

It is to be noted that the Examples include a number of molecularbiology, microbiology, immunology and biochemistry techniques consideredto be known to those skilled in the art. Disclosure of such techniquescan be found, for example, in Sambrook et al., ibid., and relatedreferences.

Example 1

This example describes the identification of high molecular weightproteins that bind to IgE from dogs known to be allergic to miteallergens.

About 5.5 grams (g) of frozen wet Dermataphagoides farinae (Der f) mites(available from Bayer Allergy, Spokane, Wash.) were homogenized in aground glass homogenizer, in either about 30 ml of phosphate bufferedsaline (PBS) or 0.1 M Tris-HCl, pH 8, each containing complete proteaseinhibitors (available from Boehringer Mannheim, Indianapolis, Ind.) toobtain a Derf crude extract. The resulting supernatants were collectedand each concentrated in a Centriprep 30 concentrator (available fromAmicon, Beverly, Ma.) by centrifugation at 16,000×g for about 30minutes. The concentrated supernatants were applied to separateSephacryl S-100 columns (2.7×70 cm; available from Pharmacia,Piscataway, N.J.) in PBS or 0.1 M Tris-HCl, pH 8, respectively. Theexcluded fractions from each column were pooled. Fractions were dialyzedagainst 10 mM Tris-HCl, pH 8, when PBS was used. The fractions wereapplied to separate Q-Sepharose columns (2.5×5 cm; available fromPharmacia). The Q-Sepharose column was pre-equilibrated in 10 mMTris-HCl, pH 8, when the fractions containing 0.1 M Tris-HCl, pH 8 wereused. Each column was sequentially eluted with about 45 ml of 10 mMTris-HCl, pH 8, then 0.1 M Tris-HCl, pH 8, then 0.2 M Tris-HCl, pH 8,then 0.3 M Tris-HCl, pH 8, then 0.4 M Tris-HCl, pH 8 and then 0.5 MTris-HCl, pH 8. Fractions were collected from each elution step. Eachfraction was analyzed by western blot for the presence of protein thatbound to IgE antibodies present in dog sera isolated from dogs known tobe allergic to mite allergens (referred to herein as mite allergic dogantisera or mite allergic antisera). Specifically, proteins contained inthe fractions were resolved by 12% Tris-glycine SDS-PAGE and thenblotted onto nitrocellulose. The blot was incubated with a pool of seraobtained from dogs known to be allergic to mite allergens, diluted 1:20,using standard buffers. The blot was incubated and then washed usingstandard procedures. The blot was then incubated with the mousemonoclonal anti-dog IgE antibody DE138 (1 mg/ml, 1:1000 dilution). Theblot was incubated and then washed using standard procedures. The blotwas then incubated with donkey anti-mouse IgG antibody conjugated tohorseradish peroxidase (1:1000 dilution; available from Jackson Labs,Maine). The presence of HRP-conjugated antibody bound to the blot wasdetected using standard techniques. An about 70-kD protein wasidentified in the 0.2 M Tris-HCl, pH 8 fraction, an about 98-kD proteinand an about 109-kD protein were identified in the 0.3 M Tris-HCl, pH 8fraction.

The fraction described above that was eluted using 0.3 M Tris-HCl, pH 8was concentrated in a Centriprep 30 concentrator and then diluted in 20mM Na—Ac, pH 5.6. The diluted fraction was then applied to a PolyCat AHPLC cation exchange column (available from PolyLC, Columbia, Md.). Thecolumn was cluted with about 10 ml of 20 mM Na—Ac, pH 5.6, and then withabout 45 ml of a linear gradient from 0 to 0.5 M NaCl in the 20 mMNa—Ac, pH 5.6 buffer at a flow rate of about 1 ml/min. Fractions werecollected from the elution procedure and assayed for the presence ofhigh molecular weight proteins using the mite allergic antisera andwestern blot protocol described above. Fractions containing the highmolecular weight proteins were pooled. Trifluoroacetic acid (TFA) wasadded to a concentration of about 0.05%. The solution was applied to aTSK-Gel TMS-250 C1 reverse phase column (available from TosoHaas,Montgomeryville, Pa.) that had been pre-equilibrated in 80% solvent Aand 20% solvent B. Solvent A was composed of about 0.05% TFA in waterand solvent B was composed of about 0.05% TFA in 90% acetonitrile inwater. The column was eluted with about 5 ml of 20% solvent B and thenwith 36 ml of a linear gradient of about 20% to about 70% solvent B at0.6 ml/min. The proteins eluted from the column were resolved by 12%Tris-Glycine PAGE. The gel was stained with Comassie blue. The stainedgel is shown in FIG. 1. Lane 1 contains Mark-12 protein molecular weightmarkers (available from Novex, San Diego, Calif.), lane 2 contains theprotein eluted from the reverse phase column, and lane 3 containsSeeBlue™ protein molecular weight markers (available from Novex). Twomajor proteins were identified in the eluant. The molecular weights ofthe proteins were determined using a BioRad™ Multi-Analyst™/PC ImageSystem (available from BioRad Corp.). The higher molecular weightprotein in lane 2 of FIG. 1 was determined to be about 109 kD, referredto herein as mite allergen protein A (mapA). The lower molecular weightprotein in lane 2 of FIG. 1 was determined to be about 98 kD, referredto herein as mite allergen protein B (mapB). The purity of the combinedproteins was greater than 85% purity, i.e., less than 15% impurities.This purified eluant is referred to herein as the D. farinae highmolecular weight map (HMW-map) composition.

Example 2

This example describes N-terminal sequencing of proteins in the D.farinae HMW-map composition.

Proteins contained in the 0.3 M Tris-HCl, pH 8 fraction obtained asdescribed above in Example 1 were resolved by SDS-PAGE using a 12%Tris-glycine polyacrylamide-SDS gel, followed by coomasie staining. Theproteins were blotted onto PVDF, stained with Coomasie R-250 anddestained using standard procedures. The proteins corresponding to theabout 98 kD and about 109 kD bands were excised and subjected separatelyto N-terminal amino acid sequencing using techniques known to thoseskilled in the art. A partial N-terminal amino acid sequence of about 14amino acids was deduced for both proteins and the sequences weredetermined to be identical. The N-terminal amino acid sequence isrepresented herein as SEQ ID NO:1, having the amino acid sequence: SerIle Lys Arg Asp His Asn Asp Tyr Ser Lys Asn Pro Met.

The proteins in the D. farinae HMW-map composition were also submittedto proteolytic cleavage in order to obtain internal amino acid sequencedata. Specifically, the D. farinae HMW-map composition was cleaved withEndoproteinase Asp-N (available from Boehringer Mannheim Biochemica,Indianapolis, Ind.) using methods standard in the art. The digestedprotein was then resolved by HPLC using the method described by Stone etal., Enzymatic Digestion of Proteins and HPLC Peptide Isolation, in APractical Guide to Protein and Peptide Purification for Microsequencing,PT Matsudaira ed., Academic Press, San Diego, Calif. Twelve proteolyticfragments were isolated, that are referred to herein as map(1), map(2),map(3), map(4), map(5), map(6), map(7), map(8), map(9), map(10), map(11)and map(12).

The N-terminal partial amino acid sequence of map(1) was determined tobe Asp Tyr Glu Tyr Pro Gly Ser Arg Leu Gly Asn Pro Lys Ala Pro Leu TyrLys Arg Pro, also denoted SEQ ID NO:2. The N-terminal partial amino acidsequence of map(2) was determined to be Asp Ile Pro His Pro Thr Asn IleHis Lys Tyr Leu Val Cys Glu Ser Val Asn Gly Gly, also denoted SEQ IDNO:3. The N-terminal partial amino acid sequence of map(3) wasdetermined to be Asp Pro Ala Lys Gly Met Ser Pro Pro Gly Phe lie Val GlyGlu Glu Gly Val Leu Ser, also denoted SEQ ID NO:4. The N-terminalpartial amino acid sequence of map(4) was determined to be Asp Glu LysAsn Ser Phe Glu Cys Ile Leu Gly Pro, also denoted SEQ ID NO:5. TheN-terminal partial amino acid sequence of map(5) was determined to beAsp Ala Phe Glu Pro His Gly Tyr Leu Leu Thr Ala Ala Val Ser Pro Gly Lys,also denoted SEQ ID NO:6. The N-terminal partial amino acid sequence ofmap(6) was determined to be Asp Lys Gin Asn Tyr Leu Ala Leu Val Arg GluLeu Lys, also denoted SEQ ID NO:7. The N-terminal partial amino acidsequence of map(7) was determined to be Asp Met Ala Gin Asn Tyr Lys TyrArg Gin Gin Phe Ile Gin Ser Val Leu Asn Asn Gly Ala Thr Arg Gin, alsodenoted SEQ ID NO:8. The N-terminal partial amino acid sequence ofmap(8) was determined to be Asp Glu Xaa Asn Val Met Xaa Tyr Val Leu TyrThr Met His Tyr Tyr Leu Asn Asn Gly Ala Thr Arg, also denoted SEQ IDNO:9, in which Xaa represents any amino acid. The N-terminal partialamino acid sequence of map(9) was determined to be Asp Lys Leu Val MetGly Val Pro Phe Tyr Gly Arg Ala Xaa Ser Ile Glu, also denoted SEQ IDNO:10, in which Xaa represents any amino acid. The N-terminal partialamino acid sequence of map(10) was determined to be Asp Ile Pro His ProThr Asn lie His Lys Tyr Leu Val Cys Glu Ser Val Asn Gly, also denotedSEQ ID NO:11. The N-terminal partial amino acid sequence of map(11) wasdetermined to be Asp Tyr Ala Lys Asn Pro Lys Arg lie Val Cys Ile Val GlyThr Glu Gly Val Leu Ser, also denoted SEQ ID NO:12. The N-terminalpartial amino acid sequence of map(12) was determined to be Asp Pro AlaLys Gly Met Ser Pro Pro Gly He Ile Val Gly Glu Glu Gly Val Leu Ser, alsodenoted SEQ ID NO:13. Since the amino acid sequences for map(1), map(2),map(3), map(4), map(5), map(6), map(7), map(8), map(9), map(10),map(11), map(12), and map(13) were generated from a mixture of mapA andmapB proteins, these sequences do not necessarily represent partialsequences of a single protein.

Example 3

This example describes the purification of a 70-kD protein that binds toIgE from dogs known to be allergic to mite allergens.

The fraction described above in Example 1 that was eluted using 0.2 MTris-HCl, pH 8 was concentrated in a Centriprep 30 concentrator and thendiluted in 20 mM Na—Ac, pH 5.6. The diluted protein was then applied toa PolyCat A HPLC cation exchange column. The column was eluted withabout 10 ml of 20 mM Na—Ac, pH 5.6, and then with about 45 ml of alinear gradient from 0 to 0.5 M NaCl in the 20 mM Na—Ac, pH 5.6 bufferat a flow rate of about 1 ml/min. Fractions were collected from theelution procedure and assayed for the presence of 70-kD protein usingthe mite allergic antisera and western blot protocol described above.Fractions containing the 70-kD protein were pooled. Trifluoroacetic acid(TFA) was added to a concentration of about 0.05%. The solution wasapplied to a TSK-Gel TMS-250 C1 reverse phase column that had beenpre-equilibrated in 80% solvent A and 20% solvent B. Solvent A wascomposed of about 0.05% TFA in water and solvent B was composed of about0.05% TFA in 90% acetonitrile in water. The column was eluted with about3 ml of 20% solvent B and then with 36 ml of a linear gradient of about20% to about 70% solvent B at 0.6 ml/min. An about 70-kD protein of >90%purity was obtained. The about 70-kD protein is referred to herein asmapC.

N-terminal sequence of a region on an SDS-PAGE corresponding to the 70kD protein (mapC) was obtained as described in Example 2. An N-terminalamino acid sequence of about 21 amino acids was deduced with an 80%confidence level, and is represented herein as SEQ ID NO:33, having thefollowing amino acid sequence: Gln Ser Arg Asp Arg Asn Asp Lys Pro TyrXaa Ile Val Lys Lys Lys Lys Lys Ala Leu Asp.

Example 4

This example describes the binding of the D. farinae HMW-map composition(i.e., containing mapA and mapB) to canine IgE in dog sera isolated fromdogs known to be allergic to mite allergens.

Multiple wells of an Immulon II microtiter plate were coated with about100 nanograms per well (ng/well) of a D. farinae HMW-map compositionisolated according to the method described above in Example 1, dilutedin CBC buffer. The plate was incubated overnight at 4° C. Followingincubation, the D. farinae HMW-map composition-containing solution wasremoved from the plate, and the plate was blotted dry. The plate wasthen blocked using about 200 μu/well of 4.0% fetal calf serum containedin phosphate buffered saline (PBS) having 0.05% Tween-20 (PBSTFCS) forabout 1 hour at room temperature. The plate was then washed four timeswith 0.05% Tween-20 in PBS (PBST) using an automatic washer (availablefrom Dynatech, Chantilly, Va.). About 100 μl/well of a 1:10 dilution inPBSTFCS of serum samples isolated from different dogs known to besensitive to mite allergens in intradermal skin tests were added to theplate. A negative control group of sera was also added to the platecomprising a combination of sera from six dogs that were raised in abarrier facility (available from Harlan Bioproducts, Indianapolis,Ind.). Some wells did not receive dog sera so that background bindinglevels could be determined. The plate was incubated for about 1 hour atroom temperature and then washed four times with PBST. About 100 μl/wellof a 1:4000 dilution of 40 μg/ml biotinylated human FcεR alpha chainprotein (produced as described in Frank et al., WO 98/23964, publishedNov. 24, 1997) contained in PBSTFCS was added. The plate was incubatedfor about 1 hour at room temperature and then washed four times withPBST. About 100 μl of about 0.25 μg/ml streptavidin conjugated tohorseradish peroxidase (available from Kirkegaard and Perry Laboratories(KPL), Gaithersburg, Md.; diluted in PBST) was added to each well thatreceived experimental or control samples. The plates were then incubatedfor about 1 hour at room temperature and washed four times with PBST.About 100 μl of TMB substrate (available from KPL), that had beenpre-warmed to room temperature, was added to each well. The plate wasthen incubated for about 10 minutes at room temperature and then about100 μl/well of Stop Solution (available from KPL) was added. Opticaldensities (O.D.) of wells were read on a Spectramax Microtiter Plate(available from Molecular Devices Inc.) reader at 450 nm within 10minutes of adding the stop solution.

The O.D. readings obtained using the negative control sample and thebackground wells were 0 O.D. Sera from 5 of 26 mite allergen sensitivedogs generated O.D. readings between about 2,000 O.D. and about 3,200O.D. Sera from 3 other mite allergen sensitive dogs generated O.D.readings between about 1,000 O.D. and 2,000 O.D. Sera from 3 other miteallergen sensitive dogs generated O.D. readings between about 500 O.D.and 1,000 O.D. Sera from 7 other mite allergen sensitive dogs generatedO.D. readings between about 200 O.D. and 500 O.D. Sera from 6 other miteallergen sensitive dogs generated O.D. readings less than 50 O.D. Thus,the results indicate that sera from dogs known to be sensitive to miteallergens contain IgE antibodies that bind specifically to the mapA andmapB proteins of the present invention.

Example 5

This example describes the binding of the 70-kD D. farinae protein tocanine IgE in dog sera isolated from dogs known to be allergic to miteallergens.

Multiple wells of an Immulon II microtiter plate were coated with about100 ng/well of 70-kD D. farinae protein (referred to herein as mapC)isolated according to the method described above in Examples 1 and 3,diluted in CBC buffer. The plate was incubated overnight at 4° C. Theplate was blocked and washed using the method described in Example 4.About 100 μl/well of a 1:10 dilution in PBSTFCS of serum samplesisolated from different dogs known to be sensitive to mite allergens inintradermal skin tests were added to the plate. Negative control sampleswere also added to the plate comprising SPF serum samples (serum fromdogs maintained in a barrier facility and therefore never exposed tomite allergens). Some wells did not receive dog sera so that backgroundbinding levels could be determined. The plate was incubated for about 1hour at room temperature and then washed four times with PBST.Biotinylated human FcεR alpha chain protein was then added and thepresence of IgE bound to the plate was detected using the methodsdescribed in Example 4.

The O.D. readings obtained using the negative control sample and thebackground wells were 0 O.D. Sera from 3 of 26 mite allergen sensitivedogs generated O.D. readings between about 1,500 O.D. and about 2,700O.D. Sera from 5 other mite allergen sensitive dogs generated O.D.readings between about 800 and about 1,500 O.D. Sera from 4 other miteallergen sensitive dogs generated O.D. readings between about 500 O.D.and about 800 O.D. Sera from 6 other mite allergen sensitive dogsgenerated O.D. readings between about 200 O.D. and 500 O.D. Sera from 8other mite allergen sensitive dogs generated O.D. readings less than 50O.D. Thus, the results indicate that sera from dogs known to besensitive to mite allergens contain IgE antibodies that bindspecifically to the mapC protein of the present invention.

Example 6

This example describes the binding of mapA, mapB or mapC proteins tofeline IgE in cat sera isolated from cats shown by in vitro testing tobe hypersensitive to mite allergens.

Multiple wells of an Immulon II microtiter plate were coated with about100 ng/well of a D. farinae HMW-map composition (isolated according tothe method described above in Example 1) and 70-kD D. farinae protein(isolated according to the method described above in Example 3). Otherwells of the plate were coated with 400 ng/well of wholeDermatophagoides pteronyssius extract (available from GreerLaboratories, Inc., Lenoir, N.C.; concentrated 8-fold prior to use) orwhole D. farinae extract (available from Miles, Inc., Elkhart, Ind.).All samples were diluted in CBC buffer. The plates were incubatedovernight at 4° C. The plates were blocked and washed using the methoddescribed in Example 4. About 100 μl/well of a 1:10 dilution in PBSTFCSof serum samples isolated from different cats known to be sensitive tomite allergens in in vitro allergen testing were added to the plate.Sera from seven control cats (#15, #16, #17, #18, #19, #20, and #21),shown not to be sensitive by in vitro test to dust mite allergens, werealso tested. Some wells did not receive cat sera so that backgroundbinding levels could be determined. The plate was incubated for about 1hour at room temperature and then washed four times with PBST.Biotinylated human FcεR alpha chain protein was then added and thepresence of IgE bound to the plate was detected using the methodsdescribed in Example 4.

The results are shown below in Table 1. All values represent O.D. valuestimes 1,000. HDM refers to cats that are sensitive to house dust miteallergens (by serological test, i.e. an ELISA to whole D. farinaeextract).

TABLE 1 Cat # HDM Whole Der p Whole Der f mapA and mapB mapC 1 + 54 173211 400 2 + 437 454 245 352 3 + 96 88 17 36 4 + 35 179 278 758 5 + 12323 0 0 6 + 2 10 0 0 7 + 84 321 439 445 8 + 125 333 611 599 9 + 2459 27371613 507 10 + 17 0 0 0 11 + 146 347 243 586 12 + 31 100 102 223 13 + 56171 267 292 14 + 121 146 163 185 15 − 0 0 0 8 16 − 0 0 0 0 17 − 0 0 0 018 − 0 0 0 0 19 − 0 0 0 0 20 − 0 0 0 0 21 − 23 0 0 0

The results indicate that sera from some of the cats known to besensitive to mite allergens contain IgE antibodies that boundspecifically to the mapA, mapB or mapC proteins of the presentinvention. In addition, some sera containing IgE that bound to the mapA,mapB or mapC proteins also contain IgE antibodies that bound to whole D.pteronyssius extract. The control sera did not contain IgE antibodiesthat bound to either the mapA, mapB or mapC proteins of the presentinvention.

Example 7

This example demonstrates the ability of the D. farinae HMW-mapcomposition to induce a hypersensitive response in dogs.

To determine whether the D. farinae HMW-map composition described inExample 1 was capable of inducing an allergic response in animalssusceptible to dust mite allergic responses, skin tests were performedon dogs that actively demonstrate clinical signs for dust mite allergy(referred to herein as atopic dogs). Normal dogs include dogs that donot show symptoms of mite allergy but may be susceptible to a miteallergic response. Each dog (i.e., 4 normal and 4 atopic dogs) wasshaved in the lateral thorax/abdominal area and intradermally injectedin different sites in that area with an about 1:50,000 dilution of D.farinae crude extract isolated by the method described in Example 1,with about 2 μg of the purified D. farinae HMW-map composition and/orwith control solutions, i.e., saline, as a negative control, and a1:1000 dilution of histamine as a positive control. All four normal dogsand all 4 atopic dogs received D. farinae whole extract. Three of thenormal dogs and 2 of the atopic dogs received the D. farinae HMW-mapcomposition. All 8 of the dogs received both the negative and positivecontrol samples. The total volume per injection was 50 microliters (μl), with the compositions and controls being diluted in saline. Theinjections were administered as single injections.

All injection sites were objectively measured in millimeters (mm) at 15minutes and scored either (+) or (−) when compared with the controlsamples. The subjective scoring was performed by Andrew Hillier, D. V.M., at Ohio State University, Columbus, Ohio. The results are shown inTable 2:

TABLE 2 Normal Normal Normal Normal Atopic Atopic Atopic Atopic Dog 1Dog 2 Dog 3 Dog 4 Dog 1 Dog 2 Dog 3 Dog 4 Whole Extract + + + − + + − −HMW map + + − n/a + − n/a n/a Neg. Control − − − − − − − −Histamine + + + + + + + + n/a = not applicable

The results indicate that the D. farinae HMW-map composition was capableof inducing an immediate hypersensitive response in dogs includingatopic dogs. Thus, the HMW-map composition is sufficiently allergenic toinduce a hypersensitive response in dogs including atopic dogs.

Table 3 describes the results of the following experiment. IgE to theHMW-map composition was measured in the serum of three groups of dogs:D. farinae allergic (HDM-AD), atopic (to other allergens) but not HDMallergic (AD), and naive dogs using ELISA. These dogs were also testedby intradermal skin test to D. farinae whole extract and to the HMW-mapcomposition.

TABLE 3 Skin test and ELISA data for D. farinae whole extract and forHMW-map composition in D. farinae-allergic, atopic but not HDM-allergic, and naive dogs. Clinical Df IDST HMW-map HMW-map Dog status1:50,000 Df ELISA IDST 1 ug ELISA 1 HDM-AD + 1968 + 2876 2 HDM-AD + 407− 954 3 HDM-AD + 3921 + 3465 4 HDM-AD + 153 + 198 5 HDM-AD + 1712 + 9976 HDM-AD + 1833 + 2006 7 HDM-AD + 4200 + 4200 8 HDM-AD + 2851 + 3559 9HDM-AD + 122 + 209 10 HDM-AD + 1627 + 566 11 HDM-AD + 1185 + 1307 12HDM-AD + 308 + 101 13 HDM-AD + 341 + 433 14 AD − 1 − 0 15 AD − 8 − 2 16AD ND 66 ND 87 17 Normal − 24 − 40 18 Normal − 53 ND 369 19 Normal − 37− 21 20 SPF beagle ND 0 ND 0 21 SPF beagle ND 6 ND 1

All dogs that were positive by ELISA for whole D. farinae extract werealso positive for the HMW-map composition allergen. Of the eight dogsthat were ELISA negative for whole D. farinae extract, 7 of 8 were alsonegative for the HMW-map composition.

Example 8

This example describes the isolation of nucleic acid molecules encodinga Der HMW-map composition of the present invention.

Der HMW-map composition nucleic acid molecules were identified andisolated as follows.

A. Preparation of a Dermatophagoides farinae cDNA Library

A Dermatophagoides farinae cDNA library was prepared as follows. TotalRNA was extracted from about 2 grams of flash frozen and pulverizedhouse dust mites, using an acid-guanidinium-phenol-chloroform methodsimilar to that described by Chomzynski et al., 1987, Anal. Biochem.162,156-159. Poly A⁺ selected RNA was separated from the total RNApreparation by oligo-dT cellulose chromatography using the mRNAPurification Kit ( available from Pharmacia Biotech, Newark, N.J.),according to the method recommended by the manufacturer. A cDNA librarywas constructed in lambda-Uni-ZAP™ XR vector (available fromStratagene), using Stratagene's ZAP-cDNA Synthesis Kit protocol.Approximately 5 μg of Poly A⁺ RNA was used to produce theDermatophadoides farinae cDNA library.

B. Preparation of PCR Primers

Further N-terminal amino acid sequence analysis was performed accordingto the methods described above in Example 2. A partial N-terminal aminoacid sequence of 34 amino acids was deduced and is represented by SEQ IDNO:24, having the amino acid sequence: Ser lie Lys Arg Asp His Asn AspTyr Ser Lys Asn Pro Met Met Ile Val Xaa Tyr Tyr Gly Gly Ser Ser Gly TyrGln Ser Xaa Lys Arg Xaa Xaa Thr (wherein “Xaa” represents any amino acidresidue). The amino acid sequences of SEQ ID NO:4 (described above inExample 2) and SEQ ID NO:24 were used to design syntheticoligonucleotide primers. Sense primer Derf1 derived from SEQ ID NO:24,having the nucleotide sequence 5′ AAA CGT GAT CAT AAY GAT TAY TCN AARAAY C 3′ (wherein Y represents C or T, R represents A or G, and Nrepresents A, C, T or G), designated SEQ ID NO:25 or sense primer Derf2,derived from SEQ ID NO:24, having the nucleotide sequence 5′ AAA CGT GATCAT AAY GAT TAY AGY AAR AAY C 3′, designated SEQ ID NO:26, were used incombination with antisense primer Derf3 derived from SEQ ID NO:4, havingthe nucleotide sequence 5′ CCT TCT TCA CCN ACR ATC AAN CC 3′, denotedSEQ ID NO:27, or antisense primer Derf4 derived from SEQ ID NO:4, havingthe nucleotide sequence 5′ CCT TCT TCA CCN ACR ATG AAN CC 3′, denotedSEQ ID NO:28.

The foregoing primers were then used to screen the Der f cDNA libraryusing standard polymerase chain reaction amplification (PCR) techniques.All attempts to identify a cDNA that hybridized to the primers failed.

C. Immunoscreening the D. farinae cDNA Library Using anti-DerHMW-mapcomposition Antibodies

Since attempts to isolate a cDNA clone using PCR methods failed, theinventors screened the D. farinae cDNA library using an antiserumproduced as follows. Protein isolated according to the method describedabove in Example 1 was used as a source of antigen to generate rabbitpolyclonal antibodies, referred to herein as anti-Der HMW-mapcomposition antibodies. The preparation of rabbit polyclonal antibodieswas carried out using standard techniques.

About 7.5 ml of Escherichia coli (XL1 Blue, O.D.₆₀₀=0.5) was incubatedwith 3.0×10⁴ pfu of phage from a Dermatophagoides farinae ZAP-cDNAlibrary (1.8×10⁹ pfu/ml), at 37° C. for 15 min and plated in 30 mlLuria-Bertani (LB) medium agar plates (150 mm). The plates wereincubated at 37° C. over night. Each plate was then overlaid with anIPTG (10 mM) treated nitrocellulose filter for about 4 hours at 37° C.The filters were then removed and washed with Tris buffered saline (pH7.5) containing 0.1% Tween (TBST), for 5 minutes. The filters wereblocked with a solution of 1% dried powder milk, 1% BSA, 2% goat serumand 0.15% gelatin, prepared in TBST, for 2 hours at room temperature.Filters were then incubated with the anti-Der HMW-map compositionantibodies at a dilution of 1:1000, contained in the above blockingsolution at 4° C., overnight. The mixture was then incubated with adonkey anti-rabbit IgG antibody conjugated to horseradish peroxidase(available from Jackson ImmunoResearch, West Grove, PN) for 2 hours atroom temperature. All of the filters were washed with blocking solutioncontained in TBST (3×15 min/wash) between each incubation. All of thefilters were then treated to a final wash in Tris buffered saline (pH7.5) for 5 minutes at room temperature. Immunocomplexed plaques wereidentified by immersing the filters into the developing solution (TMBPeroxidase Substrate/TMB Peroxidase Solution/TMB Membrane Enhancer fromKirkegaard & Perry Laboratories) at 1/1/0.1 volume ratio to produce acolor reaction. One hundred and twenty three plaques were identified and50 plaques were further plaque purified two more times under the sameimmunoscreening condition as described above.

D. PCR Screening of Purified Phage Plugs

The phage plugs identified in the foregoing immunoscreening study werethen further analyzed by PCR amplification using the primers describedabove in section 8B. DNA from the 50 plaques was amplified using amixture of the 4 primers identified by SEQ ID NO:25, SEQ ID NO:26, SEQID NO:27 and SEQ ID NO:28. PCR amplification was conducted usingstandard techniques. One resulting PCR amplification product comprised afragment of about 700 nucleotides. The PCR product was cloned into theInVitrogen, Corp., TA™ cloning vector (procedures provided by InVitrogen, Corp.) and subjected to DNA sequence analysis using standardtechniques. The phagemid from the purified phage that were determined tocontain sequences encoded in the 700-bp PCR product were rescued andsubjected to DNA sequence analysis using standard techniques.

A clone was isolated that included about a 1752-nucleotide insert,referred to herein as nDerf98₁₇₅₂. Nucleic acid sequence was obtainedusing standard techniques from nDerf98₁₇₅₂, to yield a Dermatophagoidesfarinae nucleic acid molecule named nDerf98₁₇₅₂ composed of a codingstrand having nucleic acid sequence SEQ ID NO:14 and a complementarystrand having a nucleic acid sequence SEQ ID NO:16. Translation of SEQID NO:14 suggests that nucleic acid molecule nDerf98₁₇₅₂ encodes afull-length flea protein of about 555 amino acids, referred to herein asPDerf98₅₅₅, having amino acid sequence SEQ ID NO:15, assuming an openreading frame in which the first codon spans from nucleotide 1 throughnucleotide 3 of SEQ ID NO:14 and a stop codon spanning from nucleotide1666 through nucleotide 1668 of SEQ ID NO:14. The amino acid sequence ofPDerf98₅₅₅ is encoded by the nucleic acid molecule nDerf98₁₆₆₅, having acoding strand with the nucleic acid sequence SEQ ID NO:17 and acomplementary strand with the nucleic acid sequence SEQ ID NO:19.PDerf98₅₅₅, also represented by SEQ ID NO:18, has an estimated molecularweight of about 63.2 kD and an estimated pI of about 5.33. Analysis ofSEQ ID NO:15 suggests the presence of a signal peptide spanning fromabout amino acid 1 through about amino acid 19. The proposed matureprotein, denoted herein as PDerf₅₃₆, contains about 536 amino acids, thesequence of which is represented herein as SEQ ID NO:21, and is encodedby a nucleic acid molecule referred to herein as nDerf98₁₆₀₈,represented by SEQ ID NO:20, the coding strand, and SEQ ID NO:22, thecomplementary strand. The amino acid sequence of flea PDerf98₅₃₆ (i.e.SEQ ID NO:21) predicts that PDerf98₅₃₆ has an estimated molecular weightof 61.2 kD, and an estimated pI of about 5.26.

Comparison of amino acid sequence SEQ ID NO:15 with amino acid sequencesreported in GenBank indicates that SEQ ID NO:15 showed the mosthomology, i.e., about 42% identity, with a chitinase protein fromAnopheles gambiae (GenBank accession number 2654602). Comparison ofnucleic acid sequence SEQ ID NO:17 with nucleic acid sequences reportedin GenBank indicates that SEQ ID NO:17 showed the most homology, i.e.,about 58% identity between SEQ ID NO:17 and Chelonus sp. venom chitinasemRNA (GenBank accession number U10422).

Example 9

This example describes the purification of a 60-kD protein that binds toIgE from dogs known to be allergic to mite allergens and partial aminoacid sequences derived from this 60-kD protein.

A. Purification of a 60 kD Protein

D. farinae extract was prepared and fractionated on a Sephacryl S-100column according to the methods described above in Example 1. Fractionswere collected from the Sephacryl S-100 column after the excluded peak(fractions 29 through 35) and were pooled. The pooled fractions werethen diluted 1:1 with 10 mM Tris-HCl, pH 8, and applied to a Q-sepharosecolumn and fractions obtained using the methods described above inExample 1. The fraction that eluted in 0.4 M Tris-HCl was concentratedand further purified through a TMS 250 reverse phase HPLC column usingthe methods described above in Example 1. The proteins in the fractionswere resolved by 14% Tris-glycine SDS-PAGE using similar methodsdescribed for resolution of proteins on the 12% gel in Example 1. Thestained gel is shown in FIG. 2. A protein was identified having amolecular weight of about 60 kD (FIG. 2, lane 4) of about 90% puritythat eluted at about 50% B (0.05%TA in 90% acetonitrile). The molecularweight of the denoted 60-kd protein was estimated to be 56.11 kd usingthe BioRad Multi-Analyst/PC Version 1.1 program and Mark-12 proteinmolecular weight markers. The about 60-kd protein is referred to hereinas mapD protein.

B. Partial N-terminal and Internal Sequence Obtained from the 60-kdProtein

The eluted protein from Part A, above, was blotted onto PVDF, which wasstained with Coomassie R-250 and destained using standard procedures.The protein corresponding to the about 60-kd band was excised andsubjected to N-terminal amino acid sequencing using techniques known tothose skilled in the art. A partial N-terminal amino acid sequence ofabout 25 amino acids was deduced for the protein and the amino acidsequence, represented herein as SEQ ID NO:23, was determined to be: XaaLeu Glu Pro Lys Thr Val Cys Tyr Tyr Glu Ser Trp Val His His Arg Gln GlyGlu Gly Lys Met Asp Pro (wherein Xaa refers to any amino acid).

The protein corresponding to the 60 kd region was also submitted toproteolytic cleavage in order to obtain internal amino acid sequencedata. Digestion of the 60-kd protein and reverse-phase chromatographywere carried out as described in Example 1. Four proteolytic fragmentswere isolated and sequenced, and are referred to herein as map(13),map(14), map(15), and map(16).

The N-terminal partial amino acid sequence of map(13) was determined tobe Gln Tyr Gly Val Thr Gln Ala Val Val Thr Gln ProAla, also denoted SEQID NO:29. The N-termninal partial amino acid sequence of map(14) wasdetermined to be Asp Glu Leu Leu Met Lys Ser Gly Pro Gly Pro, alsodenoted SEQ ID NO:30. The N-terminal partial amino acid sequence ofmap(15) was determined to be Asp Met Glu His Phe Thr Gln His Lys Gly AsnAla Lys Ala Met Ile Ala Val Gly Gly Ser Thr Met Ser, also denoted SEQ IDNO:31. The N-terminal partial amino acid sequence of map(16) wasdetermined to be Asp Ala Asn Glu Glu Ala Arg Ser Gln Leu Pro Glu Thr AlaMet Val Leu Ile Lys Ser Gln, denoted SEQ ID NO:32.

Example 10

This example describes the isolation and sequencing of nucleic acidmolecules encoding a portion of the D. farinae 60 kD (mapD) allergen.

A D. farinae library was prepared as described previously in Example 8.A degenerate synthetic oligonucleotide primer was designed from theN-terminal amino acid sequence deduced for D. farinae 60 kD-protein (SEQID NO:23): Primer 1, a sense primer corresponding to amino acid residuesfrom about 3 through about 11 of SEQ ID NO:23 has the sequence 5′GAACCAAAA CHGTNTGYTA YTAYG 3′, also known as SEQ ID NO:46, where Hrepresents A or C or T, N represents A or C or G or T, and Y representsC or T. PCR amplification of fragments from the D. farinae library wasconducted using standard techniques. A PCR amplification product wasgenerated using a combination of SEQ ID NO:46 (primer 1) and the M13forward universal primer 5′GTAAAACGACG GCCAGT 3′, denoted SEQ ID NO:47.

A second, nested PCR reaction was carried out on the products of thefirst PCR reaction. A synthetic oligonucleotide was synthesized thatcorresponded to a region spanning from about amino acid residue 1through amino acid residue 10 of the 60-kD protein internal amino acidsequence, SEQ ID NO:31. This primer, primer 2, has the nucleic acidsequence 5′ GATATGGAAC ATTTYACHCA ACAYAARGG 3′, denoted SEQ ID NO:48,where R represents A or G. A PCR amplification product was generatedusing the combination of primer 2, SEQ ID NO:48, and the T7 standardprimer, 5′ GTAATACGAC TCACTATAGG GC 3′, denoted SEQ ID NO:49. Theresultant PCR product was subjected to DNA sequence analysis usingstandard techniques.

The PCR product was sequenced and found to contain 510 nucleotides, andis known as nDerf60₅₁₀. The nucleotide sequence of the coding strand ofnDerf60₅₁₀ is represented herein as SEQ ID NO:43, and its complement isdenoted SEQ ID NO:45. Translation of SEQ ID NO:43 suggests thatnDerf60₅₁₀ encodes a partial D. farinae 60-kD protein of about 170 aminoacids, referred to herein as PDerf60₁₇₀, with an amino acid sequencedenoted SEQ ID NO:44, assuming an open reading frame in which the firstcodon spans from about nucleotide 1 through nucleotide 3 of SEQ IDNO:43, and the last codon spanning from about nucleotide 508 throughabout nucleotide 510 of SEQ ID NO:43. PDerf60₁₇₀ has an estimatedmolecular weight of 19.2 kD and an estimated pI of about 6.51.

Nucleic acid molecule nDerf60₅₁₀ was used as a probe to isolate anucleic acid molecule that encodes a protein corresponding to afull-length, or larger partial D. farinae 60-kD protein. Usingprocedures described previously in Example 8, the whole D. farinaelibrary was screened with the nucleic acid SEQ ID NO:43 radiolabeledwith ³²P using standard techniques. Hybridization was done in 6×SSC,5×Denhardt's solution, 0.5% SDS, 100 mg/ml ssDNA, at 55° C., for about36 hours. The filters were washed 3 times, for 30 minutes per wash, at55° C. in 2×SSC, 0.2% SDS, followed by a final wash of about 30 minutesin 0.2×SSC, 0.2% SDS.

PCR amplification was carried out on the primary phage plugs. Primer 1,denoted as SEQ ID NO:46, and T7 standard primer, denoted as SEQ IDNO:49, were used as the primers, and a PCR product was generated.Preliminary sequence analysis of this 1.6 kilobase PCR product showedthat it represents a nucleic acid sequence that contains the completesequence encoding the PDerf60 full-length protein.

Comparison of PDerf60₁₇₀, the amino acid sequence of SEQ ID NO:44, withamino acid sequences reported in GenBank indicates that PDerf60₁₇₀showed the most homology, i.e. about 39% identity, with a chitinaseprotein precursor from Aphanodidium album. (GenBank accession numberP32470). Nucleic acid sequence SEQ ID NO:43 showed no significanthomology to any of the sequences submitted to GenBank.

Example 11

This example describes the isolation of nucleic acid molecules encodingDermatophagoides pteronyssius 98 kD allergen protein.

Nucleic acid molecules with high homology to the D. farinae 98 kDallergen (map B) were isolated from a D. pteronyssius cDNA library byhybridization with a 32-P labeled cDNA encoding the D. farinae HMW-mapcomposition.

A D. pteronyssius cDNA library was prepared as follows. Total RNA wasextracted from approximately 2 grams of D. pteronyssius mites, using anacid-guanidium-phenol-chloroform method, described by Chomzynski et al.,1987, Anal. Biochem 162: pp 156-159. Poly A+ selected RNA was separatedfrom the total RNA preparation by oligo-dT cellulose chromatographyusing the mRNA Purification Kit (available from Pharmacia, Newark,N.J.), according to the method recommended by the manufacturer. A wholeD. pteronyssius cDNA library was constructed in lambda-Uni-ZAP™ XRvector (available from Stratagene, La Jolla, Calif.), using Stratagene'sZAP-cDNA Synthesis Kit protocol. Approximately 5 milligram (mg) of PolyA+ RNA was used to produce the D. pteronyssius eDNA library.

Using a modification of the protocol described in the cDNA Synthesis Kit(available from Stratagene), the whole D. pteronyssius cDNA library wasscreened, using duplicate plaque lifts, with a 32P-labeled cDNA encodingthe D. farinae 97 kcD Map B allergen, i.e. SEQ ID NO:17. Hybridizationwas done in 6×SSC (for recipe see Sambrook, et al., ibid.), 5×Denhardt'ssolution (for recipe see Sambrook, et al., ibid.), 0.5% sodium dodecylsulfate (SDS) (available from Sigma), and 100 mg/ml of single 10stranded DNA (available from Sigma), at 55° C., for about 36 hours. Thefilters were washed 3 times, for about 30 minutes per wash, at 55° C.,in 2×SSC, 0.2% SDS, followed by a final wash of about 30 minutes, at 55°C., in 0.2×SSC, 0.2% SDS. A plaque purified clone of the D. pteronyssiusnucleic acid molecule encoding the D. pteronyssius 97 kD allergen (mapB) was converted into a double stranded recombinant molecule using theExAssis™ helper phage and SOLR™ E. coli according to the in vivoexcision protocol described in the ZAP-eDNA Synthesis Kit (all availablefrom Stratagene). The plasmid containing the D. pteronyssius clone wassubjected to DNA sequence analysis using standard techniques. DNAsequence analysis, including the determination of molecular weight andisoelectric point (pI) was performed using the GCG™ program.

A clone was isolated that included an about 1621-nucleotide insert,which includes the full-length coding region, referred to herein asnDerp98₁₆₂₁, with a coding strand represented as SEQ ID NO:34 and acomplementary strand represented as SEQ ID NO:36. The apparent start andstop codons span from nucleotide 14 through nucleotide 16, and fromnucleotide 1541 through nucleotide 1543, respectively, of SEQ ID NO:34.A putative polyadenylation signal (5′AATAAA 3′) is located in a regionspanning from nucleotide 1584 to 1589 of SEQ ID NO:34.

Translation of SEQ ID NO:34 yields a protein of about 509 amino acids,denoted PDerp98₅₀₉, the amino acid sequence of which is presented as SEQID NO:35. The nucleic acid molecule consisting of the coding regionencoding PDerp98₅₀₉ is referred to herein as nDerp98₁₅₂₇, the nucleicacid sequence of which is represented as SEQ ID NO:37 (the codingstrand), and SEQ ID NO:39 (the complementary strand). The amino acidsequence of PDerp98₅₀₉, also represented herein as SEQ ID NO:38, has anestimated molecular weight of about 58.9 kD and an estimated pI of about5.61. Analysis of PDerp98₅₀₉ suggests the presence of a signal peptidespanning from about amino acid 1 through about amino acid 19. Theproposed mature protein, denoted herein as PDerp98₄₉₀, contains about490 amino acids, and is represented herein as SEQ ID NO:41. The aminoacid sequence of PDerp98₄₉₀ predicts the protein to have an estimatedmolecular weight of about 56.8 kD, and an estimated pI of about 5.49, aswell as two asparagine-linked glycosylation sites extending from aboutamino acid 115 to about amino acid 117, and extending from about aminoacid 240 to amino acid 242, respectively. The nucleic acid moleculeencoding PDerp98₄₉₀ is known as nDerp98₁₄₇₀, with a coding strandrepresented by SEQ ID NO:40 and a complementary strand represented bySEQ ID NO:42.

A BLAST search was performed as described previously. PDerp98₅₀₉, SEQ IDNO:35, showed the highest homology at the amino acid level with theManduca sexta chitinase (SwissProt accession number p36362), with abouta 34% identity. nDerp98₁₆₂₁, SEQ ID NO;34, showed the highest homologyat the nucleic acid level to Chelonus sp. chitinase (accession numberU10422), with about a 49% identity. Comparison of cDNA regionscorresponding to the coding regions for the D. farinae 98 kD allergenprotein and the cDNA regions corresponding to the coding regions for theD. pteronyssius 98 kD allergen protein shows an identity of about 84%.

Example 14

This example demonstrates the binding of the D. farinae HMW-mapcomposition to human IgE in human sera isolated from humans known to beallergic to mite allergens.

A technique called RAST, or radio-allergo-absorbent test, was usedbecause the amount of human IgE present in human sera is quite low. RASTwas essentially performed as described in Aalberse, RC et al., (1981) J.Allergy Clin Immun. 68: pp 35614 364. To calculate the unit IU/ml, astandard curve was derived by performing RAST with several dilutions ofa well-characterized chimeric human/mouse IgE monoclonal antibodyagainst Derp2, (human IgE/monoclonal anti-Derp2, following the procedureof Schuurman, et al. (1997) J Allergy Clin Immunol. 99: pp 545-550).

Briefly, 50 μg of the HMW-map composition, purified as described inExample 1, was coupled to 50 mg of CNBr-activated Sepharose 4B(available from Pharmacia, Piscataway, N.J.), according to themanufacturer's protocols. Human sera were selected (17 differentsamples, total) on the basis of a positive RAST for whole mite D.farinae extracts, a positive RAST number is greater than 1 IU/ml). Twonegative (less than 0.3 IU) control sera were also included.

To test each individual serum sample, 0.5 mg of the D. farinae HMW-mapcomposition-coupled Sepharose was incubated with 50 μl serum in a totalvolume of 300 μl of PBS-T (Phosphate-buffered saline-witty-added 0.1%volume/volume Tween-20, available from Sigma). Incubation was overnightat 27° C., with shaking. After incubation, the coupled Sepharose waswashed five times with PBS-T. Radiolabelled (¹²⁵-Iodine) sheepanti-human IgE, made by standard radioiodination protocols, (diluted inPBS-T with 4.5% bovine serum and 0.5% sheep serum, v/v) in a totalvolume of 750 μl, was added and incubated overnight at 27° C. Afterincubation, the coupled Sepharose was washed four times with PBS-T andcounted in a gamma-counter to determine the amount of radiolabeled sheepanti-human IgE bound to the HMW-map composition-coupled Sepharose. Theresults are shown in Table 4.

TABLE 4 Binding of human IgE to HMW-map composition from D. farinaeRAST, D. farinae whole RAST, HMW-map Serum number extract, IU comps'n.,IU 1445 >100 48 1456 >100 42 1458 21.1 0.5 1460 14.1 2.5 1463 37.6 0.11464 37.2 2.0 1465 14.5 0.7 1466 89.9 7.7 1468 >100 19.9 1471 31.9 0.81491 23.8 1.0 1496 25.3 3.6 1505 5.1 0.2 1523 1.0 <0.1 1529 1.2 0.7 1530(control) 0.2 <0.1 1531 (control) 0.1 <0.1

Almost 75% of patients (11 of 15) who showed sensitivity to D. farinaewhole mite extracts were sensitive to the HMW-map composition antigen,implying that the HMW-map composition antigen is a major antigen for D.farinae sensitive humans. Sensitivity to the HMW-map composition wasdefined as a RAST of greater than or equal to 0.5 IU.

Example 15

This example demonstrates that the D. farinae HMW-map compositiondescribed in Example 1 includes a glycoprotein.

About (5.4 μg) of a D. farinae HMW-map composition prepared inaccordance with Example 1 was applied to SDS PAGE and electrophoresiswas done according to standard techniques. The protein was blotted to anitrocellulose membrane according to standard techniques, andglycoprotein was detected using the DIG™ Glycine Detection Kit(available from Boehringer Mannheim, Indianapolis, Ind.), using themanufacturer's protocol. The region corresponding to the HMW-map regionshowed a positive reaction with the kit, indicating that the HMW-mapcomposition includes a glycoprotein.

Example 16

This example shows that the D. farinae HMW-map composition retains itscharacter as an allergen even when the amino acid residues are removed,both by chemical and enzymatic means. The results suggest that the mainepitope(s) could be a carbohydrate epitope including a polysaccharideattached to an N-linked or O-linked glycosylation site on the HMW-mapcomposition.

A. Protein Elimination by Chemical Means (β-elimination of Proteins)

Twelve μg (microgram) of HMW-map composition (purified as described inExample 1) was dissolved in 100 μl (microliter) of distilled deionizedwater. To this mixture was added 5 μl 10 M (molar) NaOH and 3.8 mg(milligram) NaBH₄ (available from Sigma) to give a final concentrationof 0.5 M NaOH and 1 M NaBH₄. This reaction mixture was heated at 50° C.for 30 minutes, then cooled, and 100 μl acetone was added. To thismixture, sufficient amount, i.e. approximately 150 μl , of Dowex 50 (H+)(available from Pharmacia) was added to make the solution slightlyacidic. The Dowex 50 adsorbed and removed the protein, leaving any sugarmoieties in the supernatant. The mixture was centrifuged in amicrocentrifuge and washed three times with 100 μl of water. Thecombined supernatants from the centrifugations were evaporated todryness, then washed five times from a methanol:HCl solution (1000:1v/v), evaporating to dryness after each wash, to remove salts. Themixture was dissolved in 100 μl of water, and a portion (20 μl) wasanalyzed by SDS-PAGE using standard techniques, and both Coomassie blueand Silver staining were used to determine the amount of protein in thechemically treated samples. No protein was detected by either Coomassieor Silver staining, indicating removal of protein. Any sugar moieties onthe protein would be unaffected by these conditions.

The remainder of the residue from each sample was subjected to ELISAanalysis as described in Example 4. Briefly, 100 ng of either theβ-eliminated sample or of non-β-eliminated sample of the HMW-mapcomposition was coated onto the Immulon plates, and ELISAs were carriedout as described in Example 4 with a D. farinae sensitive dog sera pool,a D. farinae sensitive cat sera pool, and various individual dog serathat are either D. farinae sensitive or not sensitive (as measured byELISA). The results are shown in Table 5.

TABLE 5 Reactivity of dog and cat sera to HMW-map composition and toβ-eliminated HMW-map composition (which is carbohydrate only)β-eliminated HMW-map, untreated HMW-map Sera used OD (carbohydrateantigen) comps'n., OD × 10⁻³ D. farinae dog pool 1233 1931 D. farinaecat pool 2837 3115 dog 1621A 15 0 dog 1621C 24 21 dog 1621S 59 420 dog1626C 23 214 dog SPF-2 16 0

Results from Table 5 indicate that the β-eliminated HMW-map compositionsample still retains the ability to bind IgE from dog and cat sera thatis sensitive to D. farinae HMW-map composition, indicating that theglycans attached to the protein constitute a major epitope of theHMW-map composition allergen protein.

B. Protein Elimination by Enzymatic Means

14 μg of HMW-map composition (purified as described in Example 1) wasdigested with 1 μg Endoproteinase K, available from Sigma, to remove theprotein moiety of the molecule. The digestion reaction took place at 56°C. for 24 hours, after which the endoproteinase in the reaction washeat-denatured in boiling water for 10 minutes.

A portion of this reaction was analyzed by SDS-PAGE using standardtechniques, and both Coomassie blue and Silver staining were used todetect the presence of protein in the enzymatically digested samples. NoHMW-map composition was detected by either Coomassie or Silver staining,indicating elimination of the HMW-map composition. Any glycan that wasattached via a glycosylation site on the protein would be unaffected bythese conditions.

The remainder of the enzymatically digested reaction was tested by ELISAin the manner described in Example 4. Briefly, 100 ng of either theproteinase-K-digested sample or of a non-digested sample of the HMW-mapcomposition was coated onto Immulon plates, and ELISAs were carried outas described in Example 4 with various individual dog sera that wereeither D. farinae sensitive or not sensitive (as measured by ELISA). Theresults are shown in Table 6.

TABLE 6 Reactivity of dog sera to HMW-map composition and toEndoproteinase-K digested HMW-map composition. OD, wells coated with D.farinae OD, wells coated with Proteinase K dog # sensitive?¹ HMW-mapcomps'n. digested HMW-map 1 yes 120 122 2 yes 1637 1561 3 yes 858 383 4yes 914 509 5 yes 277 227 6 yes 2891 2636 7 no 10 11 8 yes 4056 3880 9yes 1920 1626 10 yes 472 432 11 yes 328 213 12 yes 2913 2530 13 yes 1232984 14 yes 3153 2355 15 no 6 46 16 yes 860 339 17 yes 2429 750 18 yes1194 351 19 yes 2655 1443 20 yes 3285 1207 21 yes 2636 1240 22 yes 1097848 23 yes 1621 1408 24 yes 2113 1592 25 yes 1169 408 26 yes 4200 420027 yes 4200 4200 28 yes 3222 2932 29 yes 2468 2118 30 yes 3339 2454 31no 0 4 ¹by ELISA in a separate experiment

Results from Table 6 indicate that the proteinase-K digested HMW-mapcomposition sample still retains the ability to bind IgE from dog andcat sera that is sensitive to D. farinae HMW-map composition, suggestingthat the glycans attached to the protein constitute a major epitope onthe HMW-map composition.

Example 17

This example describes attempts to remove N-linked glycans from theHMW-map composition.

HMW-map composition (2 μg), purified as in Example 1, was digested withN-glycosidase F (available from Boehringer-Mannheim), according to themanufacturer's directions. The digestion was analyzed by SDS-PAGE andstained according to standard protocols. 2 μg Fetuin (available fromSigma) was used as a positive N-linked glycosylated protein control.Analysis of the SDS-PAGE showed that there were no apparent differencesin the molecular weights of the intact and digested map B protein. Thepositive control, fetuin, did show a reduction of molecular weight afterdigestion with N-glycosidase F. This result indicates that there are noN-linked glycans on the HMW-map composition, or alternatively that thereare only small sized N-glycans on the HMW-map composition.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

49 1 14 PRT Dermatophagoides farinae 1 Ser Ile Lys Arg Asp His Asn AspTyr Ser Lys Asn Pro Met 1 5 10 2 20 PRT Dermatophagoides farinae 2 AspTyr Glu Tyr Pro Gly Ser Arg Leu Gly Asn Pro Lys Ala Pro Leu 1 5 10 15Tyr Lys Arg Pro 20 3 20 PRT Dermatophagoides farinae 3 Asp Ile Pro HisPro Thr Asn Ile His Lys Tyr Leu Val Cys Glu Ser 1 5 10 15 Val Asn GlyGly 20 4 20 PRT Dermatophagoides farinae 4 Asp Pro Ala Lys Gly Met SerPro Pro Gly Phe Ile Val Gly Glu Glu 1 5 10 15 Gly Val Leu Ser 20 5 12PRT Dermatophagoides farinae 5 Asp Glu Lys Asn Ser Phe Glu Cys Ile LeuGly Pro 1 5 10 6 18 PRT Dermatophagoides farinae 6 Asp Ala Phe Glu ProHis Gly Tyr Leu Leu Thr Ala Ala Val Ser Pro 1 5 10 15 Gly Lys 7 13 PRTDermatophagoides farinae 7 Asp Lys Gln Asn Tyr Leu Ala Leu Val Arg GluLeu Lys 1 5 10 8 24 PRT Dermatophagoides farinae 8 Asp Met Ala Gln AsnTyr Lys Tyr Arg Gln Gln Phe Ile Gln Ser Val 1 5 10 15 Leu Asn Asn GlyAla Thr Arg Gln 20 9 23 PRT Dermatophagoides farinae At locations 3 and7, Xaa = any amino acid 9 Asp Glu Xaa Asn Val Met Xaa Tyr Val Leu TyrThr Met His Tyr Tyr 1 5 10 15 Leu Asn Asn Gly Ala Thr Arg 20 10 17 PRTDermatophagoides farinae At location 14, Xaa = any amino acid 10 Asp LysLeu Val Met Gly Val Pro Phe Tyr Gly Arg Ala Xaa Ser Ile 1 5 10 15 Glu 1119 PRT Dermatophagoides farinae 11 Asp Ile Pro His Pro Thr Asn Ile HisLys Tyr Leu Val Cys Glu Ser 1 5 10 15 Val Asn Gly 12 18 PRTDermatophagoides farinae 12 Asp Tyr Ala Lys Asn Pro Lys Arg Ile Val CysIle Val Gly Thr Glu 1 5 10 15 Gly Val 13 20 PRT Dermatophagoides farinae13 Asp Pro Ala Lys Gly Met Ser Pro Pro Gly Phe Ile Val Gly Glu Glu 1 510 15 Gly Val Leu Ser 20 14 1752 DNA Dermatophagoides farinae CDS(1)..(1665) 14 atg aaa acc ata tat gca ata ctt agt att atg gcc tgc attggc ctt 48 Met Lys Thr Ile Tyr Ala Ile Leu Ser Ile Met Ala Cys Ile GlyLeu 1 5 10 15 atg aat gca tcc atc aaa cga gat cat aat gat tat tcg aaaaat ccg 96 Met Asn Ala Ser Ile Lys Arg Asp His Asn Asp Tyr Ser Lys AsnPro 20 25 30 atg aga att gtt tgt tat gtt gga aca tgg tcc gta tat cat aaagtt 144 Met Arg Ile Val Cys Tyr Val Gly Thr Trp Ser Val Tyr His Lys Val35 40 45 gat cca tac act atc gaa gat att gat cca ttc aag tgt aca cat tta192 Asp Pro Tyr Thr Ile Glu Asp Ile Asp Pro Phe Lys Cys Thr His Leu 5055 60 atg tat ggt ttc gct aaa att gat gaa tac aaa tac aca att caa gtt240 Met Tyr Gly Phe Ala Lys Ile Asp Glu Tyr Lys Tyr Thr Ile Gln Val 6570 75 80 ttc gat cct tac caa gat gat aac cat aac tca tgg gaa aaa cgt ggt288 Phe Asp Pro Tyr Gln Asp Asp Asn His Asn Ser Trp Glu Lys Arg Gly 8590 95 tat gaa cgt ttc aac aac ttg cga ttg aag aat cca gaa tta acc acc336 Tyr Glu Arg Phe Asn Asn Leu Arg Leu Lys Asn Pro Glu Leu Thr Thr 100105 110 atg att tca ctt ggt ggt tgg tat gaa ggc tcg gaa aaa tat tcc gat384 Met Ile Ser Leu Gly Gly Trp Tyr Glu Gly Ser Glu Lys Tyr Ser Asp 115120 125 atg gct gca aat cca aca tat cgt caa caa ttc ata caa tca gtt ttg432 Met Ala Ala Asn Pro Thr Tyr Arg Gln Gln Phe Ile Gln Ser Val Leu 130135 140 gac ttt ttg caa gaa tac aag ttc gac ggt cta gat ttg gat tgg gag480 Asp Phe Leu Gln Glu Tyr Lys Phe Asp Gly Leu Asp Leu Asp Trp Glu 145150 155 160 tat cct gga tct cga ttg ggt aac ccg aaa atc gat aaa caa aactat 528 Tyr Pro Gly Ser Arg Leu Gly Asn Pro Lys Ile Asp Lys Gln Asn Tyr165 170 175 ttg gct ttg gtt aga gaa ctt aaa gac gct ttt gaa cct cat ggctac 576 Leu Ala Leu Val Arg Glu Leu Lys Asp Ala Phe Glu Pro His Gly Tyr180 185 190 ttg ttg act gct gca gta tca cca ggt aaa gac aaa atc gac cgagct 624 Leu Leu Thr Ala Ala Val Ser Pro Gly Lys Asp Lys Ile Asp Arg Ala195 200 205 tat gat atc aaa gaa ttg aac aaa ttg ttc gat tgg atg aat gtcatg 672 Tyr Asp Ile Lys Glu Leu Asn Lys Leu Phe Asp Trp Met Asn Val Met210 215 220 aca tat gat tac cac ggt gga tgg gaa aac ttt tac ggt cac aatgct 720 Thr Tyr Asp Tyr His Gly Gly Trp Glu Asn Phe Tyr Gly His Asn Ala225 230 235 240 ccg ttg tat aaa cga cca gat gaa act gat gag ttg cac acttac ttc 768 Pro Leu Tyr Lys Arg Pro Asp Glu Thr Asp Glu Leu His Thr TyrPhe 245 250 255 aat gtc aac tac acc atg cac tat tat ttg aac aat ggt gccacc aga 816 Asn Val Asn Tyr Thr Met His Tyr Tyr Leu Asn Asn Gly Ala ThrArg 260 265 270 gac aaa ttg gta atg ggt gtt cca ttc tat ggc cgt gct tggagc att 864 Asp Lys Leu Val Met Gly Val Pro Phe Tyr Gly Arg Ala Trp SerIle 275 280 285 gaa gat cga agc aaa ctc aaa ctt gga gat cca gcc aaa ggcatg tcg 912 Glu Asp Arg Ser Lys Leu Lys Leu Gly Asp Pro Ala Lys Gly MetSer 290 295 300 ccc cca ggt ttc att tct ggt gaa gaa ggt gtc ctc tca tatata gaa 960 Pro Pro Gly Phe Ile Ser Gly Glu Glu Gly Val Leu Ser Tyr IleGlu 305 310 315 320 ttg tgt caa ttg ttt caa aaa gaa gaa tgg cat atc caatac gat gaa 1008 Leu Cys Gln Leu Phe Gln Lys Glu Glu Trp His Ile Gln TyrAsp Glu 325 330 335 tat tac aat gct cca tat ggt tac aat gat aaa atc tgggtc ggt tac 1056 Tyr Tyr Asn Ala Pro Tyr Gly Tyr Asn Asp Lys Ile Trp ValGly Tyr 340 345 350 gat gat ctg gcc agt ata tca tgc aag ttg gct ttc ctgaaa gaa tta 1104 Asp Asp Leu Ala Ser Ile Ser Cys Lys Leu Ala Phe Leu LysGlu Leu 355 360 365 ggc gtt tct ggt gtc atg gtt tgg tca ttg gaa aat gatgat ttc aaa 1152 Gly Val Ser Gly Val Met Val Trp Ser Leu Glu Asn Asp AspPhe Lys 370 375 380 ggt cac tgc gga ccg aaa aat cca ttg ttg aac aaa gttcat aat atg 1200 Gly His Cys Gly Pro Lys Asn Pro Leu Leu Asn Lys Val HisAsn Met 385 390 395 400 att aat ggc gat gaa aag aac tct ttc gaa tgc attttg ggt cca agt 1248 Ile Asn Gly Asp Glu Lys Asn Ser Phe Glu Cys Ile LeuGly Pro Ser 405 410 415 aca acg aca cca act cca acg acg aca ccc aca accccg act aca acg 1296 Thr Thr Thr Pro Thr Pro Thr Thr Thr Pro Thr Thr ProThr Thr Thr 420 425 430 cca aca act cct tct ccc acc acc ccg aca aca acccct tct ccc acc 1344 Pro Thr Thr Pro Ser Pro Thr Thr Pro Thr Thr Thr ProSer Pro Thr 435 440 445 acc ccg aca aca acc cct tct ccc acc aca ccg acaaca act cct tct 1392 Thr Pro Thr Thr Thr Pro Ser Pro Thr Thr Pro Thr ThrThr Pro Ser 450 455 460 ccc acc aca cca aca cca aca aca cca aca cca gcccct aca aca tcg 1440 Pro Thr Thr Pro Thr Pro Thr Thr Pro Thr Pro Ala ProThr Thr Ser 465 470 475 480 aca cct tcg cca acc acg acc gaa cac aca agcgaa aca cca aaa tat 1488 Thr Pro Ser Pro Thr Thr Thr Glu His Thr Ser GluThr Pro Lys Tyr 485 490 495 aca acc tat gtc gat gga cat ctt atc aaa tgttac aag gaa ggt gat 1536 Thr Thr Tyr Val Asp Gly His Leu Ile Lys Cys TyrLys Glu Gly Asp 500 505 510 atc cca cat cca acc aat ata cac aaa tat ttggtc tgt gaa ttt gtt 1584 Ile Pro His Pro Thr Asn Ile His Lys Tyr Leu ValCys Glu Phe Val 515 520 525 aat ggt ggc tgg tgg gtt cat att atg ccc tgtcca ccg ggc act att 1632 Asn Gly Gly Trp Trp Val His Ile Met Pro Cys ProPro Gly Thr Ile 530 535 540 tgg tgt caa gaa aaa ttg act tgt ata ggc gaataattctgaa aaaaaaattc 1685 Trp Cys Gln Glu Lys Leu Thr Cys Ile Gly Glu545 550 555 aattaaaatt taaaattcaa tttttaatat gaaaaattca aaaaaaaaaaaaaaaaaaaa 1745 aaaaaaa 1752 15 555 PRT Dermatophagoides farinae 15 MetLys Thr Ile Tyr Ala Ile Leu Ser Ile Met Ala Cys Ile Gly Leu 1 5 10 15Met Asn Ala Ser Ile Lys Arg Asp His Asn Asp Tyr Ser Lys Asn Pro 20 25 30Met Arg Ile Val Cys Tyr Val Gly Thr Trp Ser Val Tyr His Lys Val 35 40 45Asp Pro Tyr Thr Ile Glu Asp Ile Asp Pro Phe Lys Cys Thr His Leu 50 55 60Met Tyr Gly Phe Ala Lys Ile Asp Glu Tyr Lys Tyr Thr Ile Gln Val 65 70 7580 Phe Asp Pro Tyr Gln Asp Asp Asn His Asn Ser Trp Glu Lys Arg Gly 85 9095 Tyr Glu Arg Phe Asn Asn Leu Arg Leu Lys Asn Pro Glu Leu Thr Thr 100105 110 Met Ile Ser Leu Gly Gly Trp Tyr Glu Gly Ser Glu Lys Tyr Ser Asp115 120 125 Met Ala Ala Asn Pro Thr Tyr Arg Gln Gln Phe Ile Gln Ser ValLeu 130 135 140 Asp Phe Leu Gln Glu Tyr Lys Phe Asp Gly Leu Asp Leu AspTrp Glu 145 150 155 160 Tyr Pro Gly Ser Arg Leu Gly Asn Pro Lys Ile AspLys Gln Asn Tyr 165 170 175 Leu Ala Leu Val Arg Glu Leu Lys Asp Ala PheGlu Pro His Gly Tyr 180 185 190 Leu Leu Thr Ala Ala Val Ser Pro Gly LysAsp Lys Ile Asp Arg Ala 195 200 205 Tyr Asp Ile Lys Glu Leu Asn Lys LeuPhe Asp Trp Met Asn Val Met 210 215 220 Thr Tyr Asp Tyr His Gly Gly TrpGlu Asn Phe Tyr Gly His Asn Ala 225 230 235 240 Pro Leu Tyr Lys Arg ProAsp Glu Thr Asp Glu Leu His Thr Tyr Phe 245 250 255 Asn Val Asn Tyr ThrMet His Tyr Tyr Leu Asn Asn Gly Ala Thr Arg 260 265 270 Asp Lys Leu ValMet Gly Val Pro Phe Tyr Gly Arg Ala Trp Ser Ile 275 280 285 Glu Asp ArgSer Lys Leu Lys Leu Gly Asp Pro Ala Lys Gly Met Ser 290 295 300 Pro ProGly Phe Ile Ser Gly Glu Glu Gly Val Leu Ser Tyr Ile Glu 305 310 315 320Leu Cys Gln Leu Phe Gln Lys Glu Glu Trp His Ile Gln Tyr Asp Glu 325 330335 Tyr Tyr Asn Ala Pro Tyr Gly Tyr Asn Asp Lys Ile Trp Val Gly Tyr 340345 350 Asp Asp Leu Ala Ser Ile Ser Cys Lys Leu Ala Phe Leu Lys Glu Leu355 360 365 Gly Val Ser Gly Val Met Val Trp Ser Leu Glu Asn Asp Asp PheLys 370 375 380 Gly His Cys Gly Pro Lys Asn Pro Leu Leu Asn Lys Val HisAsn Met 385 390 395 400 Ile Asn Gly Asp Glu Lys Asn Ser Phe Glu Cys IleLeu Gly Pro Ser 405 410 415 Thr Thr Thr Pro Thr Pro Thr Thr Thr Pro ThrThr Pro Thr Thr Thr 420 425 430 Pro Thr Thr Pro Ser Pro Thr Thr Pro ThrThr Thr Pro Ser Pro Thr 435 440 445 Thr Pro Thr Thr Thr Pro Ser Pro ThrThr Pro Thr Thr Thr Pro Ser 450 455 460 Pro Thr Thr Pro Thr Pro Thr ThrPro Thr Pro Ala Pro Thr Thr Ser 465 470 475 480 Thr Pro Ser Pro Thr ThrThr Glu His Thr Ser Glu Thr Pro Lys Tyr 485 490 495 Thr Thr Tyr Val AspGly His Leu Ile Lys Cys Tyr Lys Glu Gly Asp 500 505 510 Ile Pro His ProThr Asn Ile His Lys Tyr Leu Val Cys Glu Phe Val 515 520 525 Asn Gly GlyTrp Trp Val His Ile Met Pro Cys Pro Pro Gly Thr Ile 530 535 540 Trp CysGln Glu Lys Leu Thr Cys Ile Gly Glu 545 550 555 16 1752 DNADermatophagoides farinae 16 tttttttttt tttttttttt ttttttttga atttttcatattaaaaattg aattttaaat 60 tttaattgaa tttttttttc agaattattc gcctatacaagtcaattttt cttgacacca 120 aatagtgccc ggtggacagg gcataatatg aacccaccagccaccattaa caaattcaca 180 gaccaaatat ttgtgtatat tggttggatg tgggatatcaccttccttgt aacatttgat 240 aagatgtcca tcgacatagg ttgtatattt tggtgtttcgcttgtgtgtt cggtcgtggt 300 tggcgaaggt gtcgatgttg taggggctgg tgttggtgttgttggtgttg gtgtggtggg 360 agaaggagtt gttgtcggtg tggtgggaga aggggttgttgtcggggtgg tgggagaagg 420 ggttgttgtc ggggtggtgg gagaaggagt tgttggcgttgtagtcgggg ttgtgggtgt 480 cgtcgttgga gttggtgtcg ttgtacttgg acccaaaatgcattcgaaag agttcttttc 540 atcgccatta atcatattat gaactttgtt caacaatggatttttcggtc cgcagtgacc 600 tttgaaatca tcattttcca atgaccaaac catgacaccagaaacgccta attctttcag 660 gaaagccaac ttgcatgata tactggccag atcatcgtaaccgacccaga ttttatcatt 720 gtaaccatat ggagcattgt aatattcatc gtattggatatgccattctt ctttttgaaa 780 caattgacac aattctatat atgagaggac accttcttcaccagaaatga aacctggggg 840 cgacatgcct ttggctggat ctccaagttt gagtttgcttcgatcttcaa tgctccaagc 900 acggccatag aatggaacac ccattaccaa tttgtctctggtggcaccat tgttcaaata 960 atagtgcatg gtgtagttga cattgaagta agtgtgcaactcatcagttt catctggtcg 1020 tttatacaac ggagcattgt gaccgtaaaa gttttcccatccaccgtggt aatcatatgt 1080 catgacattc atccaatcga acaatttgtt caattctttgatatcataag ctcggtcgat 1140 tttgtcttta cctggtgata ctgcagcagt caacaagtagccatgaggtt caaaagcgtc 1200 tttaagttct ctaaccaaag ccaaatagtt ttgtttatcgattttcgggt tacccaatcg 1260 agatccagga tactcccaat ccaaatctag accgtcgaacttgtattctt gcaaaaagtc 1320 caaaactgat tgtatgaatt gttgacgata tgttggatttgcagccatat cggaatattt 1380 ttccgagcct tcataccaac caccaagtga aatcatggtggttaattctg gattcttcaa 1440 tcgcaagttg ttgaaacgtt cataaccacg tttttcccatgagttatggt tatcatcttg 1500 gtaaggatcg aaaacttgaa ttgtgtattt gtattcatcaattttagcga aaccatacat 1560 taaatgtgta cacttgaatg gatcaatatc ttcgatagtgtatggatcaa ctttatgata 1620 tacggaccat gttccaacat aacaaacaat tctcatcggatttttcgaat aatcattatg 1680 atctcgtttg atggatgcat tcataaggcc aatgcaggccataatactaa gtattgcata 1740 tatggttttc at 1752 17 1665 DNADermatophagoides farinae CDS (1)..(1665) 17 atg aaa acc ata tat gca atactt agt att atg gcc tgc att ggc ctt 48 Met Lys Thr Ile Tyr Ala Ile LeuSer Ile Met Ala Cys Ile Gly Leu 1 5 10 15 atg aat gca tcc atc aaa cgagat cat aat gat tat tcg aaa aat ccg 96 Met Asn Ala Ser Ile Lys Arg AspHis Asn Asp Tyr Ser Lys Asn Pro 20 25 30 atg aga att gtt tgt tat gtt ggaaca tgg tcc gta tat cat aaa gtt 144 Met Arg Ile Val Cys Tyr Val Gly ThrTrp Ser Val Tyr His Lys Val 35 40 45 gat cca tac act atc gaa gat att gatcca ttc aag tgt aca cat tta 192 Asp Pro Tyr Thr Ile Glu Asp Ile Asp ProPhe Lys Cys Thr His Leu 50 55 60 atg tat ggt ttc gct aaa att gat gaa tacaaa tac aca att caa gtt 240 Met Tyr Gly Phe Ala Lys Ile Asp Glu Tyr LysTyr Thr Ile Gln Val 65 70 75 80 ttc gat cct tac caa gat gat aac cat aactca tgg gaa aaa cgt ggt 288 Phe Asp Pro Tyr Gln Asp Asp Asn His Asn SerTrp Glu Lys Arg Gly 85 90 95 tat gaa cgt ttc aac aac ttg cga ttg aag aatcca gaa tta acc acc 336 Tyr Glu Arg Phe Asn Asn Leu Arg Leu Lys Asn ProGlu Leu Thr Thr 100 105 110 atg att tca ctt ggt ggt tgg tat gaa ggc tcggaa aaa tat tcc gat 384 Met Ile Ser Leu Gly Gly Trp Tyr Glu Gly Ser GluLys Tyr Ser Asp 115 120 125 atg gct gca aat cca aca tat cgt caa caa ttcata caa tca gtt ttg 432 Met Ala Ala Asn Pro Thr Tyr Arg Gln Gln Phe IleGln Ser Val Leu 130 135 140 gac ttt ttg caa gaa tac aag ttc gac ggt ctagat ttg gat tgg gag 480 Asp Phe Leu Gln Glu Tyr Lys Phe Asp Gly Leu AspLeu Asp Trp Glu 145 150 155 160 tat cct gga tct cga ttg ggt aac ccg aaaatc gat aaa caa aac tat 528 Tyr Pro Gly Ser Arg Leu Gly Asn Pro Lys IleAsp Lys Gln Asn Tyr 165 170 175 ttg gct ttg gtt aga gaa ctt aaa gac gctttt gaa cct cat ggc tac 576 Leu Ala Leu Val Arg Glu Leu Lys Asp Ala PheGlu Pro His Gly Tyr 180 185 190 ttg ttg act gct gca gta tca cca ggt aaagac aaa atc gac cga gct 624 Leu Leu Thr Ala Ala Val Ser Pro Gly Lys AspLys Ile Asp Arg Ala 195 200 205 tat gat atc aaa gaa ttg aac aaa ttg ttcgat tgg atg aat gtc atg 672 Tyr Asp Ile Lys Glu Leu Asn Lys Leu Phe AspTrp Met Asn Val Met 210 215 220 aca tat gat tac cac ggt gga tgg gaa aacttt tac ggt cac aat gct 720 Thr Tyr Asp Tyr His Gly Gly Trp Glu Asn PheTyr Gly His Asn Ala 225 230 235 240 ccg ttg tat aaa cga cca gat gaa actgat gag ttg cac act tac ttc 768 Pro Leu Tyr Lys Arg Pro Asp Glu Thr AspGlu Leu His Thr Tyr Phe 245 250 255 aat gtc aac tac acc atg cac tat tatttg aac aat ggt gcc acc aga 816 Asn Val Asn Tyr Thr Met His Tyr Tyr LeuAsn Asn Gly Ala Thr Arg 260 265 270 gac aaa ttg gta atg ggt gtt cca ttctat ggc cgt gct tgg agc att 864 Asp Lys Leu Val Met Gly Val Pro Phe TyrGly Arg Ala Trp Ser Ile 275 280 285 gaa gat cga agc aaa ctc aaa ctt ggagat cca gcc aaa ggc atg tcg 912 Glu Asp Arg Ser Lys Leu Lys Leu Gly AspPro Ala Lys Gly Met Ser 290 295 300 ccc cca ggt ttc att tct ggt gaa gaaggt gtc ctc tca tat ata gaa 960 Pro Pro Gly Phe Ile Ser Gly Glu Glu GlyVal Leu Ser Tyr Ile Glu 305 310 315 320 ttg tgt caa ttg ttt caa aaa gaagaa tgg cat atc caa tac gat gaa 1008 Leu Cys Gln Leu Phe Gln Lys Glu GluTrp His Ile Gln Tyr Asp Glu 325 330 335 tat tac aat gct cca tat ggt tacaat gat aaa atc tgg gtc ggt tac 1056 Tyr Tyr Asn Ala Pro Tyr Gly Tyr AsnAsp Lys Ile Trp Val Gly Tyr 340 345 350 gat gat ctg gcc agt ata tca tgcaag ttg gct ttc ctg aaa gaa tta 1104 Asp Asp Leu Ala Ser Ile Ser Cys LysLeu Ala Phe Leu Lys Glu Leu 355 360 365 ggc gtt tct ggt gtc atg gtt tggtca ttg gaa aat gat gat ttc aaa 1152 Gly Val Ser Gly Val Met Val Trp SerLeu Glu Asn Asp Asp Phe Lys 370 375 380 ggt cac tgc gga ccg aaa aat ccattg ttg aac aaa gtt cat aat atg 1200 Gly His Cys Gly Pro Lys Asn Pro LeuLeu Asn Lys Val His Asn Met 385 390 395 400 att aat ggc gat gaa aag aactct ttc gaa tgc att ttg ggt cca agt 1248 Ile Asn Gly Asp Glu Lys Asn SerPhe Glu Cys Ile Leu Gly Pro Ser 405 410 415 aca acg aca cca act cca acgacg aca ccc aca acc ccg act aca acg 1296 Thr Thr Thr Pro Thr Pro Thr ThrThr Pro Thr Thr Pro Thr Thr Thr 420 425 430 cca aca act cct tct ccc accacc ccg aca aca acc cct tct ccc acc 1344 Pro Thr Thr Pro Ser Pro Thr ThrPro Thr Thr Thr Pro Ser Pro Thr 435 440 445 acc ccg aca aca acc cct tctccc acc aca ccg aca aca act cct tct 1392 Thr Pro Thr Thr Thr Pro Ser ProThr Thr Pro Thr Thr Thr Pro Ser 450 455 460 ccc acc aca cca aca cca acaaca cca aca cca gcc cct aca aca tcg 1440 Pro Thr Thr Pro Thr Pro Thr ThrPro Thr Pro Ala Pro Thr Thr Ser 465 470 475 480 aca cct tcg cca acc acgacc gaa cac aca agc gaa aca cca aaa tat 1488 Thr Pro Ser Pro Thr Thr ThrGlu His Thr Ser Glu Thr Pro Lys Tyr 485 490 495 aca acc tat gtc gat ggacat ctt atc aaa tgt tac aag gaa ggt gat 1536 Thr Thr Tyr Val Asp Gly HisLeu Ile Lys Cys Tyr Lys Glu Gly Asp 500 505 510 atc cca cat cca acc aatata cac aaa tat ttg gtc tgt gaa ttt gtt 1584 Ile Pro His Pro Thr Asn IleHis Lys Tyr Leu Val Cys Glu Phe Val 515 520 525 aat ggt ggc tgg tgg gttcat att atg ccc tgt cca ccg ggc act att 1632 Asn Gly Gly Trp Trp Val HisIle Met Pro Cys Pro Pro Gly Thr Ile 530 535 540 tgg tgt caa gaa aaa ttgact tgt ata ggc gaa 1665 Trp Cys Gln Glu Lys Leu Thr Cys Ile Gly Glu 545550 555 18 555 PRT Dermatophagoides farinae 18 Met Lys Thr Ile Tyr AlaIle Leu Ser Ile Met Ala Cys Ile Gly Leu 1 5 10 15 Met Asn Ala Ser IleLys Arg Asp His Asn Asp Tyr Ser Lys Asn Pro 20 25 30 Met Arg Ile Val CysTyr Val Gly Thr Trp Ser Val Tyr His Lys Val 35 40 45 Asp Pro Tyr Thr IleGlu Asp Ile Asp Pro Phe Lys Cys Thr His Leu 50 55 60 Met Tyr Gly Phe AlaLys Ile Asp Glu Tyr Lys Tyr Thr Ile Gln Val 65 70 75 80 Phe Asp Pro TyrGln Asp Asp Asn His Asn Ser Trp Glu Lys Arg Gly 85 90 95 Tyr Glu Arg PheAsn Asn Leu Arg Leu Lys Asn Pro Glu Leu Thr Thr 100 105 110 Met Ile SerLeu Gly Gly Trp Tyr Glu Gly Ser Glu Lys Tyr Ser Asp 115 120 125 Met AlaAla Asn Pro Thr Tyr Arg Gln Gln Phe Ile Gln Ser Val Leu 130 135 140 AspPhe Leu Gln Glu Tyr Lys Phe Asp Gly Leu Asp Leu Asp Trp Glu 145 150 155160 Tyr Pro Gly Ser Arg Leu Gly Asn Pro Lys Ile Asp Lys Gln Asn Tyr 165170 175 Leu Ala Leu Val Arg Glu Leu Lys Asp Ala Phe Glu Pro His Gly Tyr180 185 190 Leu Leu Thr Ala Ala Val Ser Pro Gly Lys Asp Lys Ile Asp ArgAla 195 200 205 Tyr Asp Ile Lys Glu Leu Asn Lys Leu Phe Asp Trp Met AsnVal Met 210 215 220 Thr Tyr Asp Tyr His Gly Gly Trp Glu Asn Phe Tyr GlyHis Asn Ala 225 230 235 240 Pro Leu Tyr Lys Arg Pro Asp Glu Thr Asp GluLeu His Thr Tyr Phe 245 250 255 Asn Val Asn Tyr Thr Met His Tyr Tyr LeuAsn Asn Gly Ala Thr Arg 260 265 270 Asp Lys Leu Val Met Gly Val Pro PheTyr Gly Arg Ala Trp Ser Ile 275 280 285 Glu Asp Arg Ser Lys Leu Lys LeuGly Asp Pro Ala Lys Gly Met Ser 290 295 300 Pro Pro Gly Phe Ile Ser GlyGlu Glu Gly Val Leu Ser Tyr Ile Glu 305 310 315 320 Leu Cys Gln Leu PheGln Lys Glu Glu Trp His Ile Gln Tyr Asp Glu 325 330 335 Tyr Tyr Asn AlaPro Tyr Gly Tyr Asn Asp Lys Ile Trp Val Gly Tyr 340 345 350 Asp Asp LeuAla Ser Ile Ser Cys Lys Leu Ala Phe Leu Lys Glu Leu 355 360 365 Gly ValSer Gly Val Met Val Trp Ser Leu Glu Asn Asp Asp Phe Lys 370 375 380 GlyHis Cys Gly Pro Lys Asn Pro Leu Leu Asn Lys Val His Asn Met 385 390 395400 Ile Asn Gly Asp Glu Lys Asn Ser Phe Glu Cys Ile Leu Gly Pro Ser 405410 415 Thr Thr Thr Pro Thr Pro Thr Thr Thr Pro Thr Thr Pro Thr Thr Thr420 425 430 Pro Thr Thr Pro Ser Pro Thr Thr Pro Thr Thr Thr Pro Ser ProThr 435 440 445 Thr Pro Thr Thr Thr Pro Ser Pro Thr Thr Pro Thr Thr ThrPro Ser 450 455 460 Pro Thr Thr Pro Thr Pro Thr Thr Pro Thr Pro Ala ProThr Thr Ser 465 470 475 480 Thr Pro Ser Pro Thr Thr Thr Glu His Thr SerGlu Thr Pro Lys Tyr 485 490 495 Thr Thr Tyr Val Asp Gly His Leu Ile LysCys Tyr Lys Glu Gly Asp 500 505 510 Ile Pro His Pro Thr Asn Ile His LysTyr Leu Val Cys Glu Phe Val 515 520 525 Asn Gly Gly Trp Trp Val His IleMet Pro Cys Pro Pro Gly Thr Ile 530 535 540 Trp Cys Gln Glu Lys Leu ThrCys Ile Gly Glu 545 550 555 19 1665 DNA Dermatophagoides farinae 19ttcgcctata caagtcaatt tttcttgaca ccaaatagtg cccggtggac agggcataat 60atgaacccac cagccaccat taacaaattc acagaccaaa tatttgtgta tattggttgg 120atgtgggata tcaccttcct tgtaacattt gataagatgt ccatcgacat aggttgtata 180ttttggtgtt tcgcttgtgt gttcggtcgt ggttggcgaa ggtgtcgatg ttgtaggggc 240tggtgttggt gttgttggtg ttggtgtggt gggagaagga gttgttgtcg gtgtggtggg 300agaaggggtt gttgtcgggg tggtgggaga aggggttgtt gtcggggtgg tgggagaagg 360agttgttggc gttgtagtcg gggttgtggg tgtcgtcgtt ggagttggtg tcgttgtact 420tggacccaaa atgcattcga aagagttctt ttcatcgcca ttaatcatat tatgaacttt 480gttcaacaat ggatttttcg gtccgcagtg acctttgaaa tcatcatttt ccaatgacca 540aaccatgaca ccagaaacgc ctaattcttt caggaaagcc aacttgcatg atatactggc 600cagatcatcg taaccgaccc agattttatc attgtaacca tatggagcat tgtaatattc 660atcgtattgg atatgccatt cttctttttg aaacaattga cacaattcta tatatgagag 720gacaccttct tcaccagaaa tgaaacctgg gggcgacatg cctttggctg gatctccaag 780tttgagtttg cttcgatctt caatgctcca agcacggcca tagaatggaa cacccattac 840caatttgtct ctggtggcac cattgttcaa ataatagtgc atggtgtagt tgacattgaa 900gtaagtgtgc aactcatcag tttcatctgg tcgtttatac aacggagcat tgtgaccgta 960aaagttttcc catccaccgt ggtaatcata tgtcatgaca ttcatccaat cgaacaattt 1020gttcaattct ttgatatcat aagctcggtc gattttgtct ttacctggtg atactgcagc 1080agtcaacaag tagccatgag gttcaaaagc gtctttaagt tctctaacca aagccaaata 1140gttttgttta tcgattttcg ggttacccaa tcgagatcca ggatactccc aatccaaatc 1200tagaccgtcg aacttgtatt cttgcaaaaa gtccaaaact gattgtatga attgttgacg 1260atatgttgga tttgcagcca tatcggaata tttttccgag ccttcatacc aaccaccaag 1320tgaaatcatg gtggttaatt ctggattctt caatcgcaag ttgttgaaac gttcataacc 1380acgtttttcc catgagttat ggttatcatc ttggtaagga tcgaaaactt gaattgtgta 1440tttgtattca tcaattttag cgaaaccata cattaaatgt gtacacttga atggatcaat 1500atcttcgata gtgtatggat caactttatg atatacggac catgttccaa cataacaaac 1560aattctcatc ggatttttcg aataatcatt atgatctcgt ttgatggatg cattcataag 1620gccaatgcag gccataatac taagtattgc atatatggtt ttcat 1665 20 1608 DNADermatophagoides farinae CDS (1)..(1608) 20 tcc atc aaa cga gat cat aatgat tat tcg aaa aat ccg atg aga att 48 Ser Ile Lys Arg Asp His Asn AspTyr Ser Lys Asn Pro Met Arg Ile 1 5 10 15 gtt tgt tat gtt gga aca tggtcc gta tat cat aaa gtt gat cca tac 96 Val Cys Tyr Val Gly Thr Trp SerVal Tyr His Lys Val Asp Pro Tyr 20 25 30 act atc gaa gat att gat cca ttcaag tgt aca cat tta atg tat ggt 144 Thr Ile Glu Asp Ile Asp Pro Phe LysCys Thr His Leu Met Tyr Gly 35 40 45 ttc gct aaa att gat gaa tac aaa tacaca att caa gtt ttc gat cct 192 Phe Ala Lys Ile Asp Glu Tyr Lys Tyr ThrIle Gln Val Phe Asp Pro 50 55 60 tac caa gat gat aac cat aac tca tgg gaaaaa cgt ggt tat gaa cgt 240 Tyr Gln Asp Asp Asn His Asn Ser Trp Glu LysArg Gly Tyr Glu Arg 65 70 75 80 ttc aac aac ttg cga ttg aag aat cca gaatta acc acc atg att tca 288 Phe Asn Asn Leu Arg Leu Lys Asn Pro Glu LeuThr Thr Met Ile Ser 85 90 95 ctt ggt ggt tgg tat gaa ggc tcg gaa aaa tattcc gat atg gct gca 336 Leu Gly Gly Trp Tyr Glu Gly Ser Glu Lys Tyr SerAsp Met Ala Ala 100 105 110 aat cca aca tat cgt caa caa ttc ata caa tcagtt ttg gac ttt ttg 384 Asn Pro Thr Tyr Arg Gln Gln Phe Ile Gln Ser ValLeu Asp Phe Leu 115 120 125 caa gaa tac aag ttc gac ggt cta gat ttg gattgg gag tat cct gga 432 Gln Glu Tyr Lys Phe Asp Gly Leu Asp Leu Asp TrpGlu Tyr Pro Gly 130 135 140 tct cga ttg ggt aac ccg aaa atc gat aaa caaaac tat ttg gct ttg 480 Ser Arg Leu Gly Asn Pro Lys Ile Asp Lys Gln AsnTyr Leu Ala Leu 145 150 155 160 gtt aga gaa ctt aaa gac gct ttt gaa cctcat ggc tac ttg ttg act 528 Val Arg Glu Leu Lys Asp Ala Phe Glu Pro HisGly Tyr Leu Leu Thr 165 170 175 gct gca gta tca cca ggt aaa gac aaa atcgac cga gct tat gat atc 576 Ala Ala Val Ser Pro Gly Lys Asp Lys Ile AspArg Ala Tyr Asp Ile 180 185 190 aaa gaa ttg aac aaa ttg ttc gat tgg atgaat gtc atg aca tat gat 624 Lys Glu Leu Asn Lys Leu Phe Asp Trp Met AsnVal Met Thr Tyr Asp 195 200 205 tac cac ggt gga tgg gaa aac ttt tac ggtcac aat gct ccg ttg tat 672 Tyr His Gly Gly Trp Glu Asn Phe Tyr Gly HisAsn Ala Pro Leu Tyr 210 215 220 aaa cga cca gat gaa act gat gag ttg cacact tac ttc aat gtc aac 720 Lys Arg Pro Asp Glu Thr Asp Glu Leu His ThrTyr Phe Asn Val Asn 225 230 235 240 tac acc atg cac tat tat ttg aac aatggt gcc acc aga gac aaa ttg 768 Tyr Thr Met His Tyr Tyr Leu Asn Asn GlyAla Thr Arg Asp Lys Leu 245 250 255 gta atg ggt gtt cca ttc tat ggc cgtgct tgg agc att gaa gat cga 816 Val Met Gly Val Pro Phe Tyr Gly Arg AlaTrp Ser Ile Glu Asp Arg 260 265 270 agc aaa ctc aaa ctt gga gat cca gccaaa ggc atg tcg ccc cca ggt 864 Ser Lys Leu Lys Leu Gly Asp Pro Ala LysGly Met Ser Pro Pro Gly 275 280 285 ttc att tct ggt gaa gaa ggt gtc ctctca tat ata gaa ttg tgt caa 912 Phe Ile Ser Gly Glu Glu Gly Val Leu SerTyr Ile Glu Leu Cys Gln 290 295 300 ttg ttt caa aaa gaa gaa tgg cat atccaa tac gat gaa tat tac aat 960 Leu Phe Gln Lys Glu Glu Trp His Ile GlnTyr Asp Glu Tyr Tyr Asn 305 310 315 320 gct cca tat ggt tac aat gat aaaatc tgg gtc ggt tac gat gat ctg 1008 Ala Pro Tyr Gly Tyr Asn Asp Lys IleTrp Val Gly Tyr Asp Asp Leu 325 330 335 gcc agt ata tca tgc aag ttg gctttc ctg aaa gaa tta ggc gtt tct 1056 Ala Ser Ile Ser Cys Lys Leu Ala PheLeu Lys Glu Leu Gly Val Ser 340 345 350 ggt gtc atg gtt tgg tca ttg gaaaat gat gat ttc aaa ggt cac tgc 1104 Gly Val Met Val Trp Ser Leu Glu AsnAsp Asp Phe Lys Gly His Cys 355 360 365 gga ccg aaa aat cca ttg ttg aacaaa gtt cat aat atg att aat ggc 1152 Gly Pro Lys Asn Pro Leu Leu Asn LysVal His Asn Met Ile Asn Gly 370 375 380 gat gaa aag aac tct ttc gaa tgcatt ttg ggt cca agt aca acg aca 1200 Asp Glu Lys Asn Ser Phe Glu Cys IleLeu Gly Pro Ser Thr Thr Thr 385 390 395 400 cca act cca acg acg aca cccaca acc ccg act aca acg cca aca act 1248 Pro Thr Pro Thr Thr Thr Pro ThrThr Pro Thr Thr Thr Pro Thr Thr 405 410 415 cct tct ccc acc acc ccg acaaca acc cct tct ccc acc acc ccg aca 1296 Pro Ser Pro Thr Thr Pro Thr ThrThr Pro Ser Pro Thr Thr Pro Thr 420 425 430 aca acc cct tct ccc acc acaccg aca aca act cct tct ccc acc aca 1344 Thr Thr Pro Ser Pro Thr Thr ProThr Thr Thr Pro Ser Pro Thr Thr 435 440 445 cca aca cca aca aca cca acacca gcc cct aca aca tcg aca cct tcg 1392 Pro Thr Pro Thr Thr Pro Thr ProAla Pro Thr Thr Ser Thr Pro Ser 450 455 460 cca acc acg acc gaa cac acaagc gaa aca cca aaa tat aca acc tat 1440 Pro Thr Thr Thr Glu His Thr SerGlu Thr Pro Lys Tyr Thr Thr Tyr 465 470 475 480 gtc gat gga cat ctt atcaaa tgt tac aag gaa ggt gat atc cca cat 1488 Val Asp Gly His Leu Ile LysCys Tyr Lys Glu Gly Asp Ile Pro His 485 490 495 cca acc aat ata cac aaatat ttg gtc tgt gaa ttt gtt aat ggt ggc 1536 Pro Thr Asn Ile His Lys TyrLeu Val Cys Glu Phe Val Asn Gly Gly 500 505 510 tgg tgg gtt cat att atgccc tgt cca ccg ggc act att tgg tgt caa 1584 Trp Trp Val His Ile Met ProCys Pro Pro Gly Thr Ile Trp Cys Gln 515 520 525 gaa aaa ttg act tgt ataggc gaa 1608 Glu Lys Leu Thr Cys Ile Gly Glu 530 535 21 536 PRTDermatophagoides farinae 21 Ser Ile Lys Arg Asp His Asn Asp Tyr Ser LysAsn Pro Met Arg Ile 1 5 10 15 Val Cys Tyr Val Gly Thr Trp Ser Val TyrHis Lys Val Asp Pro Tyr 20 25 30 Thr Ile Glu Asp Ile Asp Pro Phe Lys CysThr His Leu Met Tyr Gly 35 40 45 Phe Ala Lys Ile Asp Glu Tyr Lys Tyr ThrIle Gln Val Phe Asp Pro 50 55 60 Tyr Gln Asp Asp Asn His Asn Ser Trp GluLys Arg Gly Tyr Glu Arg 65 70 75 80 Phe Asn Asn Leu Arg Leu Lys Asn ProGlu Leu Thr Thr Met Ile Ser 85 90 95 Leu Gly Gly Trp Tyr Glu Gly Ser GluLys Tyr Ser Asp Met Ala Ala 100 105 110 Asn Pro Thr Tyr Arg Gln Gln PheIle Gln Ser Val Leu Asp Phe Leu 115 120 125 Gln Glu Tyr Lys Phe Asp GlyLeu Asp Leu Asp Trp Glu Tyr Pro Gly 130 135 140 Ser Arg Leu Gly Asn ProLys Ile Asp Lys Gln Asn Tyr Leu Ala Leu 145 150 155 160 Val Arg Glu LeuLys Asp Ala Phe Glu Pro His Gly Tyr Leu Leu Thr 165 170 175 Ala Ala ValSer Pro Gly Lys Asp Lys Ile Asp Arg Ala Tyr Asp Ile 180 185 190 Lys GluLeu Asn Lys Leu Phe Asp Trp Met Asn Val Met Thr Tyr Asp 195 200 205 TyrHis Gly Gly Trp Glu Asn Phe Tyr Gly His Asn Ala Pro Leu Tyr 210 215 220Lys Arg Pro Asp Glu Thr Asp Glu Leu His Thr Tyr Phe Asn Val Asn 225 230235 240 Tyr Thr Met His Tyr Tyr Leu Asn Asn Gly Ala Thr Arg Asp Lys Leu245 250 255 Val Met Gly Val Pro Phe Tyr Gly Arg Ala Trp Ser Ile Glu AspArg 260 265 270 Ser Lys Leu Lys Leu Gly Asp Pro Ala Lys Gly Met Ser ProPro Gly 275 280 285 Phe Ile Ser Gly Glu Glu Gly Val Leu Ser Tyr Ile GluLeu Cys Gln 290 295 300 Leu Phe Gln Lys Glu Glu Trp His Ile Gln Tyr AspGlu Tyr Tyr Asn 305 310 315 320 Ala Pro Tyr Gly Tyr Asn Asp Lys Ile TrpVal Gly Tyr Asp Asp Leu 325 330 335 Ala Ser Ile Ser Cys Lys Leu Ala PheLeu Lys Glu Leu Gly Val Ser 340 345 350 Gly Val Met Val Trp Ser Leu GluAsn Asp Asp Phe Lys Gly His Cys 355 360 365 Gly Pro Lys Asn Pro Leu LeuAsn Lys Val His Asn Met Ile Asn Gly 370 375 380 Asp Glu Lys Asn Ser PheGlu Cys Ile Leu Gly Pro Ser Thr Thr Thr 385 390 395 400 Pro Thr Pro ThrThr Thr Pro Thr Thr Pro Thr Thr Thr Pro Thr Thr 405 410 415 Pro Ser ProThr Thr Pro Thr Thr Thr Pro Ser Pro Thr Thr Pro Thr 420 425 430 Thr ThrPro Ser Pro Thr Thr Pro Thr Thr Thr Pro Ser Pro Thr Thr 435 440 445 ProThr Pro Thr Thr Pro Thr Pro Ala Pro Thr Thr Ser Thr Pro Ser 450 455 460Pro Thr Thr Thr Glu His Thr Ser Glu Thr Pro Lys Tyr Thr Thr Tyr 465 470475 480 Val Asp Gly His Leu Ile Lys Cys Tyr Lys Glu Gly Asp Ile Pro His485 490 495 Pro Thr Asn Ile His Lys Tyr Leu Val Cys Glu Phe Val Asn GlyGly 500 505 510 Trp Trp Val His Ile Met Pro Cys Pro Pro Gly Thr Ile TrpCys Gln 515 520 525 Glu Lys Leu Thr Cys Ile Gly Glu 530 535 22 1608 DNADermatophagoides farinae 22 ttcgcctata caagtcaatt tttcttgaca ccaaatagtgcccggtggac agggcataat 60 atgaacccac cagccaccat taacaaattc acagaccaaatatttgtgta tattggttgg 120 atgtgggata tcaccttcct tgtaacattt gataagatgtccatcgacat aggttgtata 180 ttttggtgtt tcgcttgtgt gttcggtcgt ggttggcgaaggtgtcgatg ttgtaggggc 240 tggtgttggt gttgttggtg ttggtgtggt gggagaaggagttgttgtcg gtgtggtggg 300 agaaggggtt gttgtcgggg tggtgggaga aggggttgttgtcggggtgg tgggagaagg 360 agttgttggc gttgtagtcg gggttgtggg tgtcgtcgttggagttggtg tcgttgtact 420 tggacccaaa atgcattcga aagagttctt ttcatcgccattaatcatat tatgaacttt 480 gttcaacaat ggatttttcg gtccgcagtg acctttgaaatcatcatttt ccaatgacca 540 aaccatgaca ccagaaacgc ctaattcttt caggaaagccaacttgcatg atatactggc 600 cagatcatcg taaccgaccc agattttatc attgtaaccatatggagcat tgtaatattc 660 atcgtattgg atatgccatt cttctttttg aaacaattgacacaattcta tatatgagag 720 gacaccttct tcaccagaaa tgaaacctgg gggcgacatgcctttggctg gatctccaag 780 tttgagtttg cttcgatctt caatgctcca agcacggccatagaatggaa cacccattac 840 caatttgtct ctggtggcac cattgttcaa ataatagtgcatggtgtagt tgacattgaa 900 gtaagtgtgc aactcatcag tttcatctgg tcgtttatacaacggagcat tgtgaccgta 960 aaagttttcc catccaccgt ggtaatcata tgtcatgacattcatccaat cgaacaattt 1020 gttcaattct ttgatatcat aagctcggtc gattttgtctttacctggtg atactgcagc 1080 agtcaacaag tagccatgag gttcaaaagc gtctttaagttctctaacca aagccaaata 1140 gttttgttta tcgattttcg ggttacccaa tcgagatccaggatactccc aatccaaatc 1200 tagaccgtcg aacttgtatt cttgcaaaaa gtccaaaactgattgtatga attgttgacg 1260 atatgttgga tttgcagcca tatcggaata tttttccgagccttcatacc aaccaccaag 1320 tgaaatcatg gtggttaatt ctggattctt caatcgcaagttgttgaaac gttcataacc 1380 acgtttttcc catgagttat ggttatcatc ttggtaaggatcgaaaactt gaattgtgta 1440 tttgtattca tcaattttag cgaaaccata cattaaatgtgtacacttga atggatcaat 1500 atcttcgata gtgtatggat caactttatg atatacggaccatgttccaa cataacaaac 1560 aattctcatc ggatttttcg aataatcatt atgatctcgtttgatgga 1608 23 25 PRT Dermatophagoides farinae At location 1, Xaa =any amino acid 23 Xaa Leu Glu Pro Lys Thr Val Cys Tyr Tyr Glu Ser TrpVal His His 1 5 10 15 Arg Gln Gly Glu Gly Lys Met Asp Pro 20 25 24 33PRT Dermatophagoides farinae At locations, 18, 28, 31 and 32, Xaa = anyamino acid 24 Ser Ile Lys Arg Asp His Asn Asp Tyr Ser Lys Asn Pro MetMet Ile 1 5 10 15 Val Xaa Tyr Gly Gly Ser Ser Gly Tyr Gln Ser Xaa LysArg Xaa Xaa 20 25 30 Thr 25 31 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Primer 25 aaacgtgatc ataaygatta ytcnaaraayc 31 26 31 DNA Artificial Sequence Description of Artificial SequenceSynthetic Primer 26 aaacgtgatc ataaygatta yagyaaraay c 31 27 23 DNAArtificial Sequence Description of Artificial Sequence Synthetic Primer27 ccttcttcac cnacratcaa ncc 23 28 23 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Primer 28 ccttcttcaccnacratgaa ncc 23 29 13 PRT Dermatophagoides farinae 29 Gln Tyr Gly ValThr Gln Ala Val Val Thr Gln Pro Ala 1 5 10 30 11 PRT Dermatophagoidesfarinae 30 Asp Glu Leu Leu Met Lys Ser Gly Pro Gly Pro 1 5 10 31 24 PRTDermatophagoides farinae 31 Asp Met Glu His Phe Thr Gln His Lys Gly AsnAla Lys Ala Met Ile 1 5 10 15 Ala Val Gly Gly Ser Thr Met Ser 20 32 21PRT Dermatophagoides farinae 32 Asp Ala Asn Glu Glu Ala Arg Ser Gln LeuPro Glu Thr Ala Met Val 1 5 10 15 Leu Ile Lys Ser Gln 20 33 21 PRTDermatophagoides farinae 33 Gln Ser Arg Asp Arg Asn Asp Lys Pro Tyr XaaIle Val Lys Lys Lys 1 5 10 15 Lys Lys Ala Leu Asp 20 34 1621 DNADermatophagoides farinae CDS (14)..(1540) 34 agaacttatg aaa atg aaa acgaca ttt gca ttg ttt tgt ata tgg gcc 49 Met Lys Thr Thr Phe Ala Leu PheCys Ile Trp Ala 1 5 10 tgc att ggc ttg atg aat gcg gcc act aaa cga gatcac aat aat tat 97 Cys Ile Gly Leu Met Asn Ala Ala Thr Lys Arg Asp HisAsn Asn Tyr 15 20 25 tcg aaa aat cca atg cga atc gta tgt tat gtt gga acatgg tcc gtt 145 Ser Lys Asn Pro Met Arg Ile Val Cys Tyr Val Gly Thr TrpSer Val 30 35 40 tat cat aaa gtt gat cca tac aca att gaa gat att gat cctttc aaa 193 Tyr His Lys Val Asp Pro Tyr Thr Ile Glu Asp Ile Asp Pro PheLys 45 50 55 60 tgt act cat ttg atg tat ggt ttt gct aaa atc gat gaa tacaaa tac 241 Cys Thr His Leu Met Tyr Gly Phe Ala Lys Ile Asp Glu Tyr LysTyr 65 70 75 acc att caa gtt ttt gat cca ttt caa gat gat aac cat aac tcatgg 289 Thr Ile Gln Val Phe Asp Pro Phe Gln Asp Asp Asn His Asn Ser Trp80 85 90 gaa aaa cac ggg tat gaa cgt ttc aac aac ttg aga ttg aag aat cca337 Glu Lys His Gly Tyr Glu Arg Phe Asn Asn Leu Arg Leu Lys Asn Pro 95100 105 gaa ttg acc acc atg att tca ttg ggt ggt tgg tat gaa ggt tca gaa385 Glu Leu Thr Thr Met Ile Ser Leu Gly Gly Trp Tyr Glu Gly Ser Glu 110115 120 aaa tat tcg gat atg gca gcc aat cca aca tat cgt cag caa ttt gtt433 Lys Tyr Ser Asp Met Ala Ala Asn Pro Thr Tyr Arg Gln Gln Phe Val 125130 135 140 caa tca gtt ttg gac ttt ttg caa gaa tac aaa ttc gat ggc ctagat 481 Gln Ser Val Leu Asp Phe Leu Gln Glu Tyr Lys Phe Asp Gly Leu Asp145 150 155 ttg gat tgg gaa tat cct gga tca cgg tta ggc aat cct aaa atcgat 529 Leu Asp Trp Glu Tyr Pro Gly Ser Arg Leu Gly Asn Pro Lys Ile Asp160 165 170 aaa caa aac tat tta aca tta gtt aga gaa ctt aaa gag gca tttgaa 577 Lys Gln Asn Tyr Leu Thr Leu Val Arg Glu Leu Lys Glu Ala Phe Glu175 180 185 cct ttc ggc tac ttg ttg act gcc gca gta tca ccc ggt aaa gataaa 625 Pro Phe Gly Tyr Leu Leu Thr Ala Ala Val Ser Pro Gly Lys Asp Lys190 195 200 att gac gta gct tat gag ctc aaa gaa ttg aac caa ttg ttc gattgg 673 Ile Asp Val Ala Tyr Glu Leu Lys Glu Leu Asn Gln Leu Phe Asp Trp205 210 215 220 atg aat gtc atg act tat gat tac cat ggc gga tgg gaa aatgtt ttc 721 Met Asn Val Met Thr Tyr Asp Tyr His Gly Gly Trp Glu Asn ValPhe 225 230 235 ggc cat aat gct ccg ttg tat aaa cga ccc gat gaa acg gatgaa ttg 769 Gly His Asn Ala Pro Leu Tyr Lys Arg Pro Asp Glu Thr Asp GluLeu 240 245 250 cac act tac ttc aat gtc aac tac acc atg cac tat tat ttgaac aat 817 His Thr Tyr Phe Asn Val Asn Tyr Thr Met His Tyr Tyr Leu AsnAsn 255 260 265 ggc gct act cga gac aaa ctt gtt atg ggt gtt cca ttc tatggt cgt 865 Gly Ala Thr Arg Asp Lys Leu Val Met Gly Val Pro Phe Tyr GlyArg 270 275 280 gct tgg agc atc gaa gat cga agc aaa gtc aaa ctt ggc gatccg gcc 913 Ala Trp Ser Ile Glu Asp Arg Ser Lys Val Lys Leu Gly Asp ProAla 285 290 295 300 aaa ggc atg tct cct cct ggt ttt att act ggt gaa gaaggt gtt ctc 961 Lys Gly Met Ser Pro Pro Gly Phe Ile Thr Gly Glu Glu GlyVal Leu 305 310 315 tca tac atc gaa ttg tgt cag tta ttc cag aaa gaa gaatgg cat att 1009 Ser Tyr Ile Glu Leu Cys Gln Leu Phe Gln Lys Glu Glu TrpHis Ile 320 325 330 caa tac gat gaa tat tac aat gct cca tac gga tat aatgat aaa atc 1057 Gln Tyr Asp Glu Tyr Tyr Asn Ala Pro Tyr Gly Tyr Asn AspLys Ile 335 340 345 tgg gtt ggt tac gat gat ctg gct agt ata tca tgc aagttg gcc ttt 1105 Trp Val Gly Tyr Asp Asp Leu Ala Ser Ile Ser Cys Lys LeuAla Phe 350 355 360 ctc aaa gaa ttg ggc gtc tct ggc gtt atg ata tgg tcattg gaa aac 1153 Leu Lys Glu Leu Gly Val Ser Gly Val Met Ile Trp Ser LeuGlu Asn 365 370 375 380 gat gat ttc aaa ggt cat tgc gga ccg aaa tat ccattg ttg aac aaa 1201 Asp Asp Phe Lys Gly His Cys Gly Pro Lys Tyr Pro LeuLeu Asn Lys 385 390 395 gtt cac aat atg atc aat ggt gat gaa aag aac tcttac gaa tgt ctt 1249 Val His Asn Met Ile Asn Gly Asp Glu Lys Asn Ser TyrGlu Cys Leu 400 405 410 ttg ggc cca agt aca acc aca cca aca cca acc accccg tca act act 1297 Leu Gly Pro Ser Thr Thr Thr Pro Thr Pro Thr Thr ProSer Thr Thr 415 420 425 tcg act acc aca cca acg cct acc acc acc gat agcaca agc gaa aca 1345 Ser Thr Thr Thr Pro Thr Pro Thr Thr Thr Asp Ser ThrSer Glu Thr 430 435 440 cca aaa tac act acg tat att gat gga cat ttg attaaa tgc tat aaa 1393 Pro Lys Tyr Thr Thr Tyr Ile Asp Gly His Leu Ile LysCys Tyr Lys 445 450 455 460 caa ggt tat ctt cca cat cca act gat gtt cataaa tat tta gtt tgt 1441 Gln Gly Tyr Leu Pro His Pro Thr Asp Val His LysTyr Leu Val Cys 465 470 475 gaa tat att gcc aca cca aac ggt ggt tgg tgggta cac att atg gat 1489 Glu Tyr Ile Ala Thr Pro Asn Gly Gly Trp Trp ValHis Ile Met Asp 480 485 490 tgt cca aaa gga act aga tgg cac gca aca ttaaaa aat tgt att caa 1537 Cys Pro Lys Gly Thr Arg Trp His Ala Thr Leu LysAsn Cys Ile Gln 495 500 505 gaa tgatctgata tatttgtaac tgttttttgctaaatgaaat ttaaataaaa 1590 Glu ttatttgaat ccattaaaaa aaaaaaaaaa a 162135 509 PRT Dermatophagoides farinae 35 Met Lys Thr Thr Phe Ala Leu PheCys Ile Trp Ala Cys Ile Gly Leu 1 5 10 15 Met Asn Ala Ala Thr Lys ArgAsp His Asn Asn Tyr Ser Lys Asn Pro 20 25 30 Met Arg Ile Val Cys Tyr ValGly Thr Trp Ser Val Tyr His Lys Val 35 40 45 Asp Pro Tyr Thr Ile Glu AspIle Asp Pro Phe Lys Cys Thr His Leu 50 55 60 Met Tyr Gly Phe Ala Lys IleAsp Glu Tyr Lys Tyr Thr Ile Gln Val 65 70 75 80 Phe Asp Pro Phe Gln AspAsp Asn His Asn Ser Trp Glu Lys His Gly 85 90 95 Tyr Glu Arg Phe Asn AsnLeu Arg Leu Lys Asn Pro Glu Leu Thr Thr 100 105 110 Met Ile Ser Leu GlyGly Trp Tyr Glu Gly Ser Glu Lys Tyr Ser Asp 115 120 125 Met Ala Ala AsnPro Thr Tyr Arg Gln Gln Phe Val Gln Ser Val Leu 130 135 140 Asp Phe LeuGln Glu Tyr Lys Phe Asp Gly Leu Asp Leu Asp Trp Glu 145 150 155 160 TyrPro Gly Ser Arg Leu Gly Asn Pro Lys Ile Asp Lys Gln Asn Tyr 165 170 175Leu Thr Leu Val Arg Glu Leu Lys Glu Ala Phe Glu Pro Phe Gly Tyr 180 185190 Leu Leu Thr Ala Ala Val Ser Pro Gly Lys Asp Lys Ile Asp Val Ala 195200 205 Tyr Glu Leu Lys Glu Leu Asn Gln Leu Phe Asp Trp Met Asn Val Met210 215 220 Thr Tyr Asp Tyr His Gly Gly Trp Glu Asn Val Phe Gly His AsnAla 225 230 235 240 Pro Leu Tyr Lys Arg Pro Asp Glu Thr Asp Glu Leu HisThr Tyr Phe 245 250 255 Asn Val Asn Tyr Thr Met His Tyr Tyr Leu Asn AsnGly Ala Thr Arg 260 265 270 Asp Lys Leu Val Met Gly Val Pro Phe Tyr GlyArg Ala Trp Ser Ile 275 280 285 Glu Asp Arg Ser Lys Val Lys Leu Gly AspPro Ala Lys Gly Met Ser 290 295 300 Pro Pro Gly Phe Ile Thr Gly Glu GluGly Val Leu Ser Tyr Ile Glu 305 310 315 320 Leu Cys Gln Leu Phe Gln LysGlu Glu Trp His Ile Gln Tyr Asp Glu 325 330 335 Tyr Tyr Asn Ala Pro TyrGly Tyr Asn Asp Lys Ile Trp Val Gly Tyr 340 345 350 Asp Asp Leu Ala SerIle Ser Cys Lys Leu Ala Phe Leu Lys Glu Leu 355 360 365 Gly Val Ser GlyVal Met Ile Trp Ser Leu Glu Asn Asp Asp Phe Lys 370 375 380 Gly His CysGly Pro Lys Tyr Pro Leu Leu Asn Lys Val His Asn Met 385 390 395 400 IleAsn Gly Asp Glu Lys Asn Ser Tyr Glu Cys Leu Leu Gly Pro Ser 405 410 415Thr Thr Thr Pro Thr Pro Thr Thr Pro Ser Thr Thr Ser Thr Thr Thr 420 425430 Pro Thr Pro Thr Thr Thr Asp Ser Thr Ser Glu Thr Pro Lys Tyr Thr 435440 445 Thr Tyr Ile Asp Gly His Leu Ile Lys Cys Tyr Lys Gln Gly Tyr Leu450 455 460 Pro His Pro Thr Asp Val His Lys Tyr Leu Val Cys Glu Tyr IleAla 465 470 475 480 Thr Pro Asn Gly Gly Trp Trp Val His Ile Met Asp CysPro Lys Gly 485 490 495 Thr Arg Trp His Ala Thr Leu Lys Asn Cys Ile GlnGlu 500 505 36 1621 DNA Dermatophagoides farinae 36 ttttttttttttttttaatg gattcaaata attttattta aatttcattt agcaaaaaac 60 agttacaaatatatcagatc attcttgaat acaatttttt aatgttgcgt gccatctagt 120 tccttttggacaatccataa tgtgtaccca ccaaccaccg tttggtgtgg caatatattc 180 acaaactaaatatttatgaa catcagttgg atgtggaaga taaccttgtt tatagcattt 240 aatcaaatgtccatcaatat acgtagtgta ttttggtgtt tcgcttgtgc tatcggtggt 300 ggtaggcgttggtgtggtag tcgaagtagt tgacggggtg gttggtgttg gtgtggttgt 360 acttgggcccaaaagacatt cgtaagagtt cttttcatca ccattgatca tattgtgaac 420 tttgttcaacaatggatatt tcggtccgca atgacctttg aaatcatcgt tttccaatga 480 ccatatcataacgccagaga cgcccaattc tttgagaaag gccaacttgc atgatatact 540 agccagatcatcgtaaccaa cccagatttt atcattatat ccgtatggag cattgtaata 600 ttcatcgtattgaatatgcc attcttcttt ctggaataac tgacacaatt cgatgtatga 660 gagaacaccttcttcaccag taataaaacc aggaggagac atgcctttgg ccggatcgcc 720 aagtttgactttgcttcgat cttcgatgct ccaagcacga ccatagaatg gaacacccat 780 aacaagtttgtctcgagtag cgccattgtt caaataatag tgcatggtgt agttgacatt 840 gaagtaagtgtgcaattcat ccgtttcatc gggtcgttta tacaacggag cattatggcc 900 gaaaacattttcccatccgc catggtaatc ataagtcatg acattcatcc aatcgaacaa 960 ttggttcaattctttgagct cataagctac gtcaatttta tctttaccgg gtgatactgc 1020 ggcagtcaacaagtagccga aaggttcaaa tgcctcttta agttctctaa ctaatgttaa 1080 atagttttgtttatcgattt taggattgcc taaccgtgat ccaggatatt cccaatccaa 1140 atctaggccatcgaatttgt attcttgcaa aaagtccaaa actgattgaa caaattgctg 1200 acgatatgttggattggctg ccatatccga atatttttct gaaccttcat accaaccacc 1260 caatgaaatcatggtggtca attctggatt cttcaatctc aagttgttga aacgttcata 1320 cccgtgtttttcccatgagt tatggttatc atcttgaaat ggatcaaaaa cttgaatggt 1380 gtatttgtattcatcgattt tagcaaaacc atacatcaaa tgagtacatt tgaaaggatc 1440 aatatcttcaattgtgtatg gatcaacttt atgataaacg gaccatgttc caacataaca 1500 tacgattcgcattggatttt tcgaataatt attgtgatct cgtttagtgg ccgcattcat 1560 caagccaatgcaggcccata tacaaaacaa tgcaaatgtc gttttcattt tcataagttc 1620 t 1621 371527 DNA Dermatophagoides farinae CDS (1)..(1527) 37 atg aaa acg aca tttgca ttg ttt tgt ata tgg gcc tgc att ggc ttg 48 Met Lys Thr Thr Phe AlaLeu Phe Cys Ile Trp Ala Cys Ile Gly Leu 1 5 10 15 atg aat gcg gcc actaaa cga gat cac aat aat tat tcg aaa aat cca 96 Met Asn Ala Ala Thr LysArg Asp His Asn Asn Tyr Ser Lys Asn Pro 20 25 30 atg cga atc gta tgt tatgtt gga aca tgg tcc gtt tat cat aaa gtt 144 Met Arg Ile Val Cys Tyr ValGly Thr Trp Ser Val Tyr His Lys Val 35 40 45 gat cca tac aca att gaa gatatt gat cct ttc aaa tgt act cat ttg 192 Asp Pro Tyr Thr Ile Glu Asp IleAsp Pro Phe Lys Cys Thr His Leu 50 55 60 atg tat ggt ttt gct aaa atc gatgaa tac aaa tac acc att caa gtt 240 Met Tyr Gly Phe Ala Lys Ile Asp GluTyr Lys Tyr Thr Ile Gln Val 65 70 75 80 ttt gat cca ttt caa gat gat aaccat aac tca tgg gaa aaa cac ggg 288 Phe Asp Pro Phe Gln Asp Asp Asn HisAsn Ser Trp Glu Lys His Gly 85 90 95 tat gaa cgt ttc aac aac ttg aga ttgaag aat cca gaa ttg acc acc 336 Tyr Glu Arg Phe Asn Asn Leu Arg Leu LysAsn Pro Glu Leu Thr Thr 100 105 110 atg att tca ttg ggt ggt tgg tat gaaggt tca gaa aaa tat tcg gat 384 Met Ile Ser Leu Gly Gly Trp Tyr Glu GlySer Glu Lys Tyr Ser Asp 115 120 125 atg gca gcc aat cca aca tat cgt cagcaa ttt gtt caa tca gtt ttg 432 Met Ala Ala Asn Pro Thr Tyr Arg Gln GlnPhe Val Gln Ser Val Leu 130 135 140 gac ttt ttg caa gaa tac aaa ttc gatggc cta gat ttg gat tgg gaa 480 Asp Phe Leu Gln Glu Tyr Lys Phe Asp GlyLeu Asp Leu Asp Trp Glu 145 150 155 160 tat cct gga tca cgg tta ggc aatcct aaa atc gat aaa caa aac tat 528 Tyr Pro Gly Ser Arg Leu Gly Asn ProLys Ile Asp Lys Gln Asn Tyr 165 170 175 tta aca tta gtt aga gaa ctt aaagag gca ttt gaa cct ttc ggc tac 576 Leu Thr Leu Val Arg Glu Leu Lys GluAla Phe Glu Pro Phe Gly Tyr 180 185 190 ttg ttg act gcc gca gta tca cccggt aaa gat aaa att gac gta gct 624 Leu Leu Thr Ala Ala Val Ser Pro GlyLys Asp Lys Ile Asp Val Ala 195 200 205 tat gag ctc aaa gaa ttg aac caattg ttc gat tgg atg aat gtc atg 672 Tyr Glu Leu Lys Glu Leu Asn Gln LeuPhe Asp Trp Met Asn Val Met 210 215 220 act tat gat tac cat ggc gga tgggaa aat gtt ttc ggc cat aat gct 720 Thr Tyr Asp Tyr His Gly Gly Trp GluAsn Val Phe Gly His Asn Ala 225 230 235 240 ccg ttg tat aaa cga ccc gatgaa acg gat gaa ttg cac act tac ttc 768 Pro Leu Tyr Lys Arg Pro Asp GluThr Asp Glu Leu His Thr Tyr Phe 245 250 255 aat gtc aac tac acc atg cactat tat ttg aac aat ggc gct act cga 816 Asn Val Asn Tyr Thr Met His TyrTyr Leu Asn Asn Gly Ala Thr Arg 260 265 270 gac aaa ctt gtt atg ggt gttcca ttc tat ggt cgt gct tgg agc atc 864 Asp Lys Leu Val Met Gly Val ProPhe Tyr Gly Arg Ala Trp Ser Ile 275 280 285 gaa gat cga agc aaa gtc aaactt ggc gat ccg gcc aaa ggc atg tct 912 Glu Asp Arg Ser Lys Val Lys LeuGly Asp Pro Ala Lys Gly Met Ser 290 295 300 cct cct ggt ttt att act ggtgaa gaa ggt gtt ctc tca tac atc gaa 960 Pro Pro Gly Phe Ile Thr Gly GluGlu Gly Val Leu Ser Tyr Ile Glu 305 310 315 320 ttg tgt cag tta ttc cagaaa gaa gaa tgg cat att caa tac gat gaa 1008 Leu Cys Gln Leu Phe Gln LysGlu Glu Trp His Ile Gln Tyr Asp Glu 325 330 335 tat tac aat gct cca tacgga tat aat gat aaa atc tgg gtt ggt tac 1056 Tyr Tyr Asn Ala Pro Tyr GlyTyr Asn Asp Lys Ile Trp Val Gly Tyr 340 345 350 gat gat ctg gct agt atatca tgc aag ttg gcc ttt ctc aaa gaa ttg 1104 Asp Asp Leu Ala Ser Ile SerCys Lys Leu Ala Phe Leu Lys Glu Leu 355 360 365 ggc gtc tct ggc gtt atgata tgg tca ttg gaa aac gat gat ttc aaa 1152 Gly Val Ser Gly Val Met IleTrp Ser Leu Glu Asn Asp Asp Phe Lys 370 375 380 ggt cat tgc gga ccg aaatat cca ttg ttg aac aaa gtt cac aat atg 1200 Gly His Cys Gly Pro Lys TyrPro Leu Leu Asn Lys Val His Asn Met 385 390 395 400 atc aat ggt gat gaaaag aac tct tac gaa tgt ctt ttg ggc cca agt 1248 Ile Asn Gly Asp Glu LysAsn Ser Tyr Glu Cys Leu Leu Gly Pro Ser 405 410 415 aca acc aca cca acacca acc acc ccg tca act act tcg act acc aca 1296 Thr Thr Thr Pro Thr ProThr Thr Pro Ser Thr Thr Ser Thr Thr Thr 420 425 430 cca acg cct acc accacc gat agc aca agc gaa aca cca aaa tac act 1344 Pro Thr Pro Thr Thr ThrAsp Ser Thr Ser Glu Thr Pro Lys Tyr Thr 435 440 445 acg tat att gat ggacat ttg att aaa tgc tat aaa caa ggt tat ctt 1392 Thr Tyr Ile Asp Gly HisLeu Ile Lys Cys Tyr Lys Gln Gly Tyr Leu 450 455 460 cca cat cca act gatgtt cat aaa tat tta gtt tgt gaa tat att gcc 1440 Pro His Pro Thr Asp ValHis Lys Tyr Leu Val Cys Glu Tyr Ile Ala 465 470 475 480 aca cca aac ggtggt tgg tgg gta cac att atg gat tgt cca aaa gga 1488 Thr Pro Asn Gly GlyTrp Trp Val His Ile Met Asp Cys Pro Lys Gly 485 490 495 act aga tgg cacgca aca tta aaa aat tgt att caa gaa 1527 Thr Arg Trp His Ala Thr Leu LysAsn Cys Ile Gln Glu 500 505 38 509 PRT Dermatophagoides farinae 38 MetLys Thr Thr Phe Ala Leu Phe Cys Ile Trp Ala Cys Ile Gly Leu 1 5 10 15Met Asn Ala Ala Thr Lys Arg Asp His Asn Asn Tyr Ser Lys Asn Pro 20 25 30Met Arg Ile Val Cys Tyr Val Gly Thr Trp Ser Val Tyr His Lys Val 35 40 45Asp Pro Tyr Thr Ile Glu Asp Ile Asp Pro Phe Lys Cys Thr His Leu 50 55 60Met Tyr Gly Phe Ala Lys Ile Asp Glu Tyr Lys Tyr Thr Ile Gln Val 65 70 7580 Phe Asp Pro Phe Gln Asp Asp Asn His Asn Ser Trp Glu Lys His Gly 85 9095 Tyr Glu Arg Phe Asn Asn Leu Arg Leu Lys Asn Pro Glu Leu Thr Thr 100105 110 Met Ile Ser Leu Gly Gly Trp Tyr Glu Gly Ser Glu Lys Tyr Ser Asp115 120 125 Met Ala Ala Asn Pro Thr Tyr Arg Gln Gln Phe Val Gln Ser ValLeu 130 135 140 Asp Phe Leu Gln Glu Tyr Lys Phe Asp Gly Leu Asp Leu AspTrp Glu 145 150 155 160 Tyr Pro Gly Ser Arg Leu Gly Asn Pro Lys Ile AspLys Gln Asn Tyr 165 170 175 Leu Thr Leu Val Arg Glu Leu Lys Glu Ala PheGlu Pro Phe Gly Tyr 180 185 190 Leu Leu Thr Ala Ala Val Ser Pro Gly LysAsp Lys Ile Asp Val Ala 195 200 205 Tyr Glu Leu Lys Glu Leu Asn Gln LeuPhe Asp Trp Met Asn Val Met 210 215 220 Thr Tyr Asp Tyr His Gly Gly TrpGlu Asn Val Phe Gly His Asn Ala 225 230 235 240 Pro Leu Tyr Lys Arg ProAsp Glu Thr Asp Glu Leu His Thr Tyr Phe 245 250 255 Asn Val Asn Tyr ThrMet His Tyr Tyr Leu Asn Asn Gly Ala Thr Arg 260 265 270 Asp Lys Leu ValMet Gly Val Pro Phe Tyr Gly Arg Ala Trp Ser Ile 275 280 285 Glu Asp ArgSer Lys Val Lys Leu Gly Asp Pro Ala Lys Gly Met Ser 290 295 300 Pro ProGly Phe Ile Thr Gly Glu Glu Gly Val Leu Ser Tyr Ile Glu 305 310 315 320Leu Cys Gln Leu Phe Gln Lys Glu Glu Trp His Ile Gln Tyr Asp Glu 325 330335 Tyr Tyr Asn Ala Pro Tyr Gly Tyr Asn Asp Lys Ile Trp Val Gly Tyr 340345 350 Asp Asp Leu Ala Ser Ile Ser Cys Lys Leu Ala Phe Leu Lys Glu Leu355 360 365 Gly Val Ser Gly Val Met Ile Trp Ser Leu Glu Asn Asp Asp PheLys 370 375 380 Gly His Cys Gly Pro Lys Tyr Pro Leu Leu Asn Lys Val HisAsn Met 385 390 395 400 Ile Asn Gly Asp Glu Lys Asn Ser Tyr Glu Cys LeuLeu Gly Pro Ser 405 410 415 Thr Thr Thr Pro Thr Pro Thr Thr Pro Ser ThrThr Ser Thr Thr Thr 420 425 430 Pro Thr Pro Thr Thr Thr Asp Ser Thr SerGlu Thr Pro Lys Tyr Thr 435 440 445 Thr Tyr Ile Asp Gly His Leu Ile LysCys Tyr Lys Gln Gly Tyr Leu 450 455 460 Pro His Pro Thr Asp Val His LysTyr Leu Val Cys Glu Tyr Ile Ala 465 470 475 480 Thr Pro Asn Gly Gly TrpTrp Val His Ile Met Asp Cys Pro Lys Gly 485 490 495 Thr Arg Trp His AlaThr Leu Lys Asn Cys Ile Gln Glu 500 505 39 1527 DNA Dermatophagoidesfarinae 39 ttcttgaata caatttttta atgttgcgtg ccatctagtt ccttttggacaatccataat 60 gtgtacccac caaccaccgt ttggtgtggc aatatattca caaactaaatatttatgaac 120 atcagttgga tgtggaagat aaccttgttt atagcattta atcaaatgtccatcaatata 180 cgtagtgtat tttggtgttt cgcttgtgct atcggtggtg gtaggcgttggtgtggtagt 240 cgaagtagtt gacggggtgg ttggtgttgg tgtggttgta cttgggcccaaaagacattc 300 gtaagagttc ttttcatcac cattgatcat attgtgaact ttgttcaacaatggatattt 360 cggtccgcaa tgacctttga aatcatcgtt ttccaatgac catatcataacgccagagac 420 gcccaattct ttgagaaagg ccaacttgca tgatatacta gccagatcatcgtaaccaac 480 ccagatttta tcattatatc cgtatggagc attgtaatat tcatcgtattgaatatgcca 540 ttcttctttc tggaataact gacacaattc gatgtatgag agaacaccttcttcaccagt 600 aataaaacca ggaggagaca tgcctttggc cggatcgcca agtttgactttgcttcgatc 660 ttcgatgctc caagcacgac catagaatgg aacacccata acaagtttgtctcgagtagc 720 gccattgttc aaataatagt gcatggtgta gttgacattg aagtaagtgtgcaattcatc 780 cgtttcatcg ggtcgtttat acaacggagc attatggccg aaaacattttcccatccgcc 840 atggtaatca taagtcatga cattcatcca atcgaacaat tggttcaattctttgagctc 900 ataagctacg tcaattttat ctttaccggg tgatactgcg gcagtcaacaagtagccgaa 960 aggttcaaat gcctctttaa gttctctaac taatgttaaa tagttttgtttatcgatttt 1020 aggattgcct aaccgtgatc caggatattc ccaatccaaa tctaggccatcgaatttgta 1080 ttcttgcaaa aagtccaaaa ctgattgaac aaattgctga cgatatgttggattggctgc 1140 catatccgaa tatttttctg aaccttcata ccaaccaccc aatgaaatcatggtggtcaa 1200 ttctggattc ttcaatctca agttgttgaa acgttcatac ccgtgtttttcccatgagtt 1260 atggttatca tcttgaaatg gatcaaaaac ttgaatggtg tatttgtattcatcgatttt 1320 agcaaaacca tacatcaaat gagtacattt gaaaggatca atatcttcaattgtgtatgg 1380 atcaacttta tgataaacgg accatgttcc aacataacat acgattcgcattggattttt 1440 cgaataatta ttgtgatctc gtttagtggc cgcattcatc aagccaatgcaggcccatat 1500 acaaaacaat gcaaatgtcg ttttcat 1527 40 1470 DNADermatophagoides farinae CDS (1)..(1470) 40 gcc act aaa cga gat cac aataat tat tcg aaa aat cca atg cga atc 48 Ala Thr Lys Arg Asp His Asn AsnTyr Ser Lys Asn Pro Met Arg Ile 1 5 10 15 gta tgt tat gtt gga aca tggtcc gtt tat cat aaa gtt gat cca tac 96 Val Cys Tyr Val Gly Thr Trp SerVal Tyr His Lys Val Asp Pro Tyr 20 25 30 aca att gaa gat att gat cct ttcaaa tgt act cat ttg atg tat ggt 144 Thr Ile Glu Asp Ile Asp Pro Phe LysCys Thr His Leu Met Tyr Gly 35 40 45 ttt gct aaa atc gat gaa tac aaa tacacc att caa gtt ttt gat cca 192 Phe Ala Lys Ile Asp Glu Tyr Lys Tyr ThrIle Gln Val Phe Asp Pro 50 55 60 ttt caa gat gat aac cat aac tca tgg gaaaaa cac ggg tat gaa cgt 240 Phe Gln Asp Asp Asn His Asn Ser Trp Glu LysHis Gly Tyr Glu Arg 65 70 75 80 ttc aac aac ttg aga ttg aag aat cca gaattg acc acc atg att tca 288 Phe Asn Asn Leu Arg Leu Lys Asn Pro Glu LeuThr Thr Met Ile Ser 85 90 95 ttg ggt ggt tgg tat gaa ggt tca gaa aaa tattcg gat atg gca gcc 336 Leu Gly Gly Trp Tyr Glu Gly Ser Glu Lys Tyr SerAsp Met Ala Ala 100 105 110 aat cca aca tat cgt cag caa ttt gtt caa tcagtt ttg gac ttt ttg 384 Asn Pro Thr Tyr Arg Gln Gln Phe Val Gln Ser ValLeu Asp Phe Leu 115 120 125 caa gaa tac aaa ttc gat ggc cta gat ttg gattgg gaa tat cct gga 432 Gln Glu Tyr Lys Phe Asp Gly Leu Asp Leu Asp TrpGlu Tyr Pro Gly 130 135 140 tca cgg tta ggc aat cct aaa atc gat aaa caaaac tat tta aca tta 480 Ser Arg Leu Gly Asn Pro Lys Ile Asp Lys Gln AsnTyr Leu Thr Leu 145 150 155 160 gtt aga gaa ctt aaa gag gca ttt gaa cctttc ggc tac ttg ttg act 528 Val Arg Glu Leu Lys Glu Ala Phe Glu Pro PheGly Tyr Leu Leu Thr 165 170 175 gcc gca gta tca ccc ggt aaa gat aaa attgac gta gct tat gag ctc 576 Ala Ala Val Ser Pro Gly Lys Asp Lys Ile AspVal Ala Tyr Glu Leu 180 185 190 aaa gaa ttg aac caa ttg ttc gat tgg atgaat gtc atg act tat gat 624 Lys Glu Leu Asn Gln Leu Phe Asp Trp Met AsnVal Met Thr Tyr Asp 195 200 205 tac cat ggc gga tgg gaa aat gtt ttc ggccat aat gct ccg ttg tat 672 Tyr His Gly Gly Trp Glu Asn Val Phe Gly HisAsn Ala Pro Leu Tyr 210 215 220 aaa cga ccc gat gaa acg gat gaa ttg cacact tac ttc aat gtc aac 720 Lys Arg Pro Asp Glu Thr Asp Glu Leu His ThrTyr Phe Asn Val Asn 225 230 235 240 tac acc atg cac tat tat ttg aac aatggc gct act cga gac aaa ctt 768 Tyr Thr Met His Tyr Tyr Leu Asn Asn GlyAla Thr Arg Asp Lys Leu 245 250 255 gtt atg ggt gtt cca ttc tat ggt cgtgct tgg agc atc gaa gat cga 816 Val Met Gly Val Pro Phe Tyr Gly Arg AlaTrp Ser Ile Glu Asp Arg 260 265 270 agc aaa gtc aaa ctt ggc gat ccg gccaaa ggc atg tct cct cct ggt 864 Ser Lys Val Lys Leu Gly Asp Pro Ala LysGly Met Ser Pro Pro Gly 275 280 285 ttt att act ggt gaa gaa ggt gtt ctctca tac atc gaa ttg tgt cag 912 Phe Ile Thr Gly Glu Glu Gly Val Leu SerTyr Ile Glu Leu Cys Gln 290 295 300 tta ttc cag aaa gaa gaa tgg cat attcaa tac gat gaa tat tac aat 960 Leu Phe Gln Lys Glu Glu Trp His Ile GlnTyr Asp Glu Tyr Tyr Asn 305 310 315 320 gct cca tac gga tat aat gat aaaatc tgg gtt ggt tac gat gat ctg 1008 Ala Pro Tyr Gly Tyr Asn Asp Lys IleTrp Val Gly Tyr Asp Asp Leu 325 330 335 gct agt ata tca tgc aag ttg gccttt ctc aaa gaa ttg ggc gtc tct 1056 Ala Ser Ile Ser Cys Lys Leu Ala PheLeu Lys Glu Leu Gly Val Ser 340 345 350 ggc gtt atg ata tgg tca ttg gaaaac gat gat ttc aaa ggt cat tgc 1104 Gly Val Met Ile Trp Ser Leu Glu AsnAsp Asp Phe Lys Gly His Cys 355 360 365 gga ccg aaa tat cca ttg ttg aacaaa gtt cac aat atg atc aat ggt 1152 Gly Pro Lys Tyr Pro Leu Leu Asn LysVal His Asn Met Ile Asn Gly 370 375 380 gat gaa aag aac tct tac gaa tgtctt ttg ggc cca agt aca acc aca 1200 Asp Glu Lys Asn Ser Tyr Glu Cys LeuLeu Gly Pro Ser Thr Thr Thr 385 390 395 400 cca aca cca acc acc ccg tcaact act tcg act acc aca cca acg cct 1248 Pro Thr Pro Thr Thr Pro Ser ThrThr Ser Thr Thr Thr Pro Thr Pro 405 410 415 acc acc acc gat agc aca agcgaa aca cca aaa tac act acg tat att 1296 Thr Thr Thr Asp Ser Thr Ser GluThr Pro Lys Tyr Thr Thr Tyr Ile 420 425 430 gat gga cat ttg att aaa tgctat aaa caa ggt tat ctt cca cat cca 1344 Asp Gly His Leu Ile Lys Cys TyrLys Gln Gly Tyr Leu Pro His Pro 435 440 445 act gat gtt cat aaa tat ttagtt tgt gaa tat att gcc aca cca aac 1392 Thr Asp Val His Lys Tyr Leu ValCys Glu Tyr Ile Ala Thr Pro Asn 450 455 460 ggt ggt tgg tgg gta cac attatg gat tgt cca aaa gga act aga tgg 1440 Gly Gly Trp Trp Val His Ile MetAsp Cys Pro Lys Gly Thr Arg Trp 465 470 475 480 cac gca aca tta aaa aattgt att caa gaa 1470 His Ala Thr Leu Lys Asn Cys Ile Gln Glu 485 490 41490 PRT Dermatophagoides farinae 41 Ala Thr Lys Arg Asp His Asn Asn TyrSer Lys Asn Pro Met Arg Ile 1 5 10 15 Val Cys Tyr Val Gly Thr Trp SerVal Tyr His Lys Val Asp Pro Tyr 20 25 30 Thr Ile Glu Asp Ile Asp Pro PheLys Cys Thr His Leu Met Tyr Gly 35 40 45 Phe Ala Lys Ile Asp Glu Tyr LysTyr Thr Ile Gln Val Phe Asp Pro 50 55 60 Phe Gln Asp Asp Asn His Asn SerTrp Glu Lys His Gly Tyr Glu Arg 65 70 75 80 Phe Asn Asn Leu Arg Leu LysAsn Pro Glu Leu Thr Thr Met Ile Ser 85 90 95 Leu Gly Gly Trp Tyr Glu GlySer Glu Lys Tyr Ser Asp Met Ala Ala 100 105 110 Asn Pro Thr Tyr Arg GlnGln Phe Val Gln Ser Val Leu Asp Phe Leu 115 120 125 Gln Glu Tyr Lys PheAsp Gly Leu Asp Leu Asp Trp Glu Tyr Pro Gly 130 135 140 Ser Arg Leu GlyAsn Pro Lys Ile Asp Lys Gln Asn Tyr Leu Thr Leu 145 150 155 160 Val ArgGlu Leu Lys Glu Ala Phe Glu Pro Phe Gly Tyr Leu Leu Thr 165 170 175 AlaAla Val Ser Pro Gly Lys Asp Lys Ile Asp Val Ala Tyr Glu Leu 180 185 190Lys Glu Leu Asn Gln Leu Phe Asp Trp Met Asn Val Met Thr Tyr Asp 195 200205 Tyr His Gly Gly Trp Glu Asn Val Phe Gly His Asn Ala Pro Leu Tyr 210215 220 Lys Arg Pro Asp Glu Thr Asp Glu Leu His Thr Tyr Phe Asn Val Asn225 230 235 240 Tyr Thr Met His Tyr Tyr Leu Asn Asn Gly Ala Thr Arg AspLys Leu 245 250 255 Val Met Gly Val Pro Phe Tyr Gly Arg Ala Trp Ser IleGlu Asp Arg 260 265 270 Ser Lys Val Lys Leu Gly Asp Pro Ala Lys Gly MetSer Pro Pro Gly 275 280 285 Phe Ile Thr Gly Glu Glu Gly Val Leu Ser TyrIle Glu Leu Cys Gln 290 295 300 Leu Phe Gln Lys Glu Glu Trp His Ile GlnTyr Asp Glu Tyr Tyr Asn 305 310 315 320 Ala Pro Tyr Gly Tyr Asn Asp LysIle Trp Val Gly Tyr Asp Asp Leu 325 330 335 Ala Ser Ile Ser Cys Lys LeuAla Phe Leu Lys Glu Leu Gly Val Ser 340 345 350 Gly Val Met Ile Trp SerLeu Glu Asn Asp Asp Phe Lys Gly His Cys 355 360 365 Gly Pro Lys Tyr ProLeu Leu Asn Lys Val His Asn Met Ile Asn Gly 370 375 380 Asp Glu Lys AsnSer Tyr Glu Cys Leu Leu Gly Pro Ser Thr Thr Thr 385 390 395 400 Pro ThrPro Thr Thr Pro Ser Thr Thr Ser Thr Thr Thr Pro Thr Pro 405 410 415 ThrThr Thr Asp Ser Thr Ser Glu Thr Pro Lys Tyr Thr Thr Tyr Ile 420 425 430Asp Gly His Leu Ile Lys Cys Tyr Lys Gln Gly Tyr Leu Pro His Pro 435 440445 Thr Asp Val His Lys Tyr Leu Val Cys Glu Tyr Ile Ala Thr Pro Asn 450455 460 Gly Gly Trp Trp Val His Ile Met Asp Cys Pro Lys Gly Thr Arg Trp465 470 475 480 His Ala Thr Leu Lys Asn Cys Ile Gln Glu 485 490 42 1470DNA Dermatophagoides farinae 42 ttcttgaata caatttttta atgttgcgtgccatctagtt ccttttggac aatccataat 60 gtgtacccac caaccaccgt ttggtgtggcaatatattca caaactaaat atttatgaac 120 atcagttgga tgtggaagat aaccttgtttatagcattta atcaaatgtc catcaatata 180 cgtagtgtat tttggtgttt cgcttgtgctatcggtggtg gtaggcgttg gtgtggtagt 240 cgaagtagtt gacggggtgg ttggtgttggtgtggttgta cttgggccca aaagacattc 300 gtaagagttc ttttcatcac cattgatcatattgtgaact ttgttcaaca atggatattt 360 cggtccgcaa tgacctttga aatcatcgttttccaatgac catatcataa cgccagagac 420 gcccaattct ttgagaaagg ccaacttgcatgatatacta gccagatcat cgtaaccaac 480 ccagatttta tcattatatc cgtatggagcattgtaatat tcatcgtatt gaatatgcca 540 ttcttctttc tggaataact gacacaattcgatgtatgag agaacacctt cttcaccagt 600 aataaaacca ggaggagaca tgcctttggccggatcgcca agtttgactt tgcttcgatc 660 ttcgatgctc caagcacgac catagaatggaacacccata acaagtttgt ctcgagtagc 720 gccattgttc aaataatagt gcatggtgtagttgacattg aagtaagtgt gcaattcatc 780 cgtttcatcg ggtcgtttat acaacggagcattatggccg aaaacatttt cccatccgcc 840 atggtaatca taagtcatga cattcatccaatcgaacaat tggttcaatt ctttgagctc 900 ataagctacg tcaattttat ctttaccgggtgatactgcg gcagtcaaca agtagccgaa 960 aggttcaaat gcctctttaa gttctctaactaatgttaaa tagttttgtt tatcgatttt 1020 aggattgcct aaccgtgatc caggatattcccaatccaaa tctaggccat cgaatttgta 1080 ttcttgcaaa aagtccaaaa ctgattgaacaaattgctga cgatatgttg gattggctgc 1140 catatccgaa tatttttctg aaccttcataccaaccaccc aatgaaatca tggtggtcaa 1200 ttctggattc ttcaatctca agttgttgaaacgttcatac ccgtgttttt cccatgagtt 1260 atggttatca tcttgaaatg gatcaaaaacttgaatggtg tatttgtatt catcgatttt 1320 agcaaaacca tacatcaaat gagtacatttgaaaggatca atatcttcaa ttgtgtatgg 1380 atcaacttta tgataaacgg accatgttccaacataacat acgattcgca ttggattttt 1440 cgaataatta ttgtgatctc gtttagtggc1470 43 510 DNA Dermatophagoides farinae CDS (1)..(510) 43 gat atg gaacat ttt aca caa cat aag ggc aac gcc aaa gcc atg atc 48 Asp Met Glu HisPhe Thr Gln His Lys Gly Asn Ala Lys Ala Met Ile 1 5 10 15 gcc gtc ggtggt tcg act atg tcc gat caa ttt tcc aag act gca gcg 96 Ala Val Gly GlySer Thr Met Ser Asp Gln Phe Ser Lys Thr Ala Ala 20 25 30 gta gaa cat tatcgg gaa acg ttt gtt gtt agc aca gtt gat ctt atg 144 Val Glu His Tyr ArgGlu Thr Phe Val Val Ser Thr Val Asp Leu Met 35 40 45 act cgt tat ggt ttcgat ggt gtc atg att gat tgg tct ggc atg caa 192 Thr Arg Tyr Gly Phe AspGly Val Met Ile Asp Trp Ser Gly Met Gln 50 55 60 gcc aaa gat agt gat aatttc att aaa ttg ttg gac aaa ttc gac gaa 240 Ala Lys Asp Ser Asp Asn PheIle Lys Leu Leu Asp Lys Phe Asp Glu 65 70 75 80 aag ttt gct cac acc tcgttt gtg atg ggt gtt acc ttg ccg gca acg 288 Lys Phe Ala His Thr Ser PheVal Met Gly Val Thr Leu Pro Ala Thr 85 90 95 atc gca tca tac gat aac tataac att cct gcc atc tcc aac tat gtc 336 Ile Ala Ser Tyr Asp Asn Tyr AsnIle Pro Ala Ile Ser Asn Tyr Val 100 105 110 gat ttt atg aac gtg ctt agtctg gat tac act gga tca tgg gcc cat 384 Asp Phe Met Asn Val Leu Ser LeuAsp Tyr Thr Gly Ser Trp Ala His 115 120 125 acg gtc ggt cat gct tct ccgttt cct gaa caa ctc aaa acg cta gaa 432 Thr Val Gly His Ala Ser Pro PhePro Glu Gln Leu Lys Thr Leu Glu 130 135 140 gct tac cac aaa cga ggc gctcca cgt cat aag atg gtc atg gct gta 480 Ala Tyr His Lys Arg Gly Ala ProArg His Lys Met Val Met Ala Val 145 150 155 160 cca ttt tat gca cgt acctgg att ctc gag 510 Pro Phe Tyr Ala Arg Thr Trp Ile Leu Glu 165 170 44170 PRT Dermatophagoides farinae 44 Asp Met Glu His Phe Thr Gln His LysGly Asn Ala Lys Ala Met Ile 1 5 10 15 Ala Val Gly Gly Ser Thr Met SerAsp Gln Phe Ser Lys Thr Ala Ala 20 25 30 Val Glu His Tyr Arg Glu Thr PheVal Val Ser Thr Val Asp Leu Met 35 40 45 Thr Arg Tyr Gly Phe Asp Gly ValMet Ile Asp Trp Ser Gly Met Gln 50 55 60 Ala Lys Asp Ser Asp Asn Phe IleLys Leu Leu Asp Lys Phe Asp Glu 65 70 75 80 Lys Phe Ala His Thr Ser PheVal Met Gly Val Thr Leu Pro Ala Thr 85 90 95 Ile Ala Ser Tyr Asp Asn TyrAsn Ile Pro Ala Ile Ser Asn Tyr Val 100 105 110 Asp Phe Met Asn Val LeuSer Leu Asp Tyr Thr Gly Ser Trp Ala His 115 120 125 Thr Val Gly His AlaSer Pro Phe Pro Glu Gln Leu Lys Thr Leu Glu 130 135 140 Ala Tyr His LysArg Gly Ala Pro Arg His Lys Met Val Met Ala Val 145 150 155 160 Pro PheTyr Ala Arg Thr Trp Ile Leu Glu 165 170 45 510 DNA Dermatophagoidesfarinae 45 ctcgagaatc caggtacgtg cataaaatgg tacagccatg accatcttatgacgtggagc 60 gcctcgtttg tggtaagctt ctagcgtttt gagttgttca ggaaacggagaagcatgacc 120 gaccgtatgg gcccatgatc cagtgtaatc cagactaagc acgttcataaaatcgacata 180 gttggagatg gcaggaatgt tatagttatc gtatgatgcg atcgttgccggcaaggtaac 240 acccatcaca aacgaggtgt gagcaaactt ttcgtcgaat ttgtccaacaatttaatgaa 300 attatcacta tctttggctt gcatgccaga ccaatcaatc atgacaccatcgaaaccata 360 acgagtcata agatcaactg tgctaacaac aaacgtttcc cgataatgttctaccgctgc 420 agtcttggaa aattgatcgg acatagtcga accaccgacg gcgatcatggctttggcgtt 480 gcccttatgt tgtgtaaaat gttccatatc 510 46 25 DNA ArtificialSequence Description of Artificial Sequence Synthetic Primer 46gaaccaaaaa chgtntgyta ytayg 25 47 17 DNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Primer 47 gtaaaacgac ggccagt 17 48 29DNA Artificial Sequence Description of Artificial Sequence SyntheticPrimer 48 gatatggaac atttyachca acayaargg 29 49 22 DNA ArtificialSequence Description of Artificial Sequence Synthetic Primer 49gtaatacgac tcactatagg gc 22

What is claimed is:
 1. An isolated nucleic acid molecule comprising anucleic acid sequence selected from the group consisting of SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:45 and complementsthereof.
 2. The method of claim 1, wherein said therapeutic compositionfurther comprises a component selected from the group consisting of anexcipient, an adjuvant and a carrier.
 3. An isolated nucleic acidmolecule encoding a Der HMW-map protein comprising a nucleic acidsequence selected from the group consisting of SEQ ID NO:14, SEQ IDNO:17, SEQ ID NO:20, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:45.
 4. Arecombinant vector comprising the insolated nucleic acid molecule as setforth in claim 3, operatively linked to a transcription controlsequence.
 5. The recombinant vector as set forth in claim 4, whereinsaid recombinant vector is a viral vector.
 6. A insolated host cellcomprising the isolated nucleic acid molecule as set forth in claim 3.7. A method to produce a mite allergenic protein, said methodcomprising: (a) obtaining a host cell having the isolated nucleic acidmolecule of claim 3 operatively linked to a transcriptional controlsequence; (b) culturing the host cell under conditions to promoteexpressions of the mite allergenic protein from the isolated nucleicacid; and (c) recovering said mite allergenic protein expressed by thetransformed cell.
 8. An isolated nucleic acid molecule comprising anucleic acid sequence that encodes a protein having an amino acidsequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO 15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, and SEQ ID NO:44 or acomplement of said nucleic acid sequence.
 9. A recombinant vectorcomprising the isolated nucleic acid molecule as set forth in claim 8operatively linked to a transcription control sequence.
 10. A insolatedhost cell comprising the isolated nucleic acid molecule as set forth inclaim
 8. 11. A method to produce a mite allergenic protein, said methodcomprising: (a) obtaining a host cell having the isolated nucleic acidmolecule of claim 8 operatively linked to a transcriptional controlsequence; (b) culturing the host cell under conditions to promote theexpression of the mite allergenic protein from the isolated nucleicacid; and (e) recovering said mite allergenic protein expressed by thetransformed cell.
 12. The isolated nucleic acid molecule of claim 8,wherein said amino acid sequence is selected from the group consistingof SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO5, SEQID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, and SEQ ID NO:13.
 13. The insolated nucleic acidmolecule of claim 8, wherein said amino acid sequence is SEQ ID NO:15.14. The isolated nucleic acid molecule of claim 8, wherein said aminoacid sequence is SEQ ID NO:18.
 15. The isolated nucleic acid molecule ofclaim 8, wherein said amino acid sequence is SEQ ID NO:21.
 16. Theisolated nucleic acid molecule of claim 8, wherein said amino acidsequence is SEQ ID NO:23.
 17. The isolated nucleic acid molecule ofclaim 8, wherein said amino acid sequence is SEQ ID NO:24.
 18. Theisolated nucleic acid molecule of claim 8, wherein said amino acidsequence is selected from the group consisting of SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33.
 19. The isolatednucleic acid molecule of claim 8, wherein said amino acid sequence isSEQ ID NO:35.
 20. The isolated nucleic acid molecule of claim 8, whereinsaid amino acid sequence is SEQ ID NO:38.
 21. The isolated nucleic acidmolecule of claim 8, wherein said amino acid sequence is SEQ ID NO:41.22. The isolated nucleic acid molecule of claim 8, wherein said aminoacid sequence is SEQ ID NO:44.